Non-obese Diabetic Research Articles

Type 1 diabetes and parasite infection: An exploratory study in NOD mice.

Microorganisms have long been suspected to influence the outcome of immune-related syndromes, particularly autoimmune diseases. Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing beta cells of pancreatic islets, causing high glycemia levels. Genetics is part of its aetiology, but environmental factors, particularly infectious microorganisms, also play a role. Bacteria, viruses, and parasites influence the outcome of T1D in mice and humans. We used nonobese diabetic (NOD) mice, which spontaneously develop T1D, to investigate the influence of a parasitic infection, leishmaniasis. Leishmania amazonensis is an intracellular eukaryotic parasite that replicates predominantly in macrophages and is responsible for cutaneous leishmaniasis. The implication of Th1 immune responses in T1D and leishmaniasis led us to study this parasite in the NOD mouse model. We previously constructed osteopontin knockout mice with a NOD genetic background and demonstrated that this protein plays a role in the T1D phenotype. In addition, osteopontin (OPN) has been found to play a role in the immune response to various infectious microorganisms and to be implicated in other autoimmune conditions, such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in mice. We present herein data demonstrating the role of OPN in the response to Leishmania in NOD mice and the influence of this parasitic infection on T1D. This exploratory study aimed to investigate the environmental infectious component of the autoimmune response, including Th1 immunity, which is common to both T1D and leishmaniasis.

Apigenin alleviates Sjögren’s syndrome-induced salivary gland epithelial cell ferroptosis via ERα signaling-mediated regulation of the ATF3/SLC7A1l axis

BackgroundIn Sjögren’s syndrome (SS)—an autoimmune disease characterized by dry mouth and eyes—salivary gland epithelial cells (SGECs) undergo ferroptosis, which disrupts their integrity and impairs saliva secretion. Apigenin, a phytoestrogen, is known to activate estrogen signalling and alleviate xerostomia in ovariectomized mice; however, its effect on SGEC survival and function in SS remains unclear. We hypothesized that apigenin alleviates SS symptoms and progression by inhibiting ferroptosis in SGECs and aimed to elucidate the underlying mechanism. MethodsApigenin (50 mg/kg) was orally gavaged to non-obese diabetic (NOD)/LtJ female mice (SS model); changes in SS functional indicators were analyzed using mRNA sequencing and bioinformatic analyses of submandibular glands. Interferon-gamma (IFN-γ)-stimulated SGECs were used to model SS in vitro; SGEC activity and aquaporin 5 (AQP5) expression were analyzed. Immunohistochemical staining, transmission electron microscopy, RT-qPCR, western blotting and other methods were used to verify the mechanisms. ResultsApigenin significantly increased salivary secretion and AQP5 expression while inhibiting ferroptosis and immune infiltration in NOD mouse submandibular glands. The oxidative stress gene ATF3 was upregulated and GPX4 was downregulated in NOD mice compared to that in control group (ICR mice); however, apigenin reversed this effect. IFN-γ treatment downregulated AQP5, SLC7A11, and GPX4 expression while promoting ATF3 expression and ferroptosis, which was mitigated by apigenin. ATF3 knockdown increased SLC7A11 and GPX4 expression, inhibiting SS and ferroptosis. Furthermore, apigenin inhibited ferroptosis in SGECs through ESR1 binding to ATF3. ConclusionApigenin alleviates SS by regulating SGEC ferroptosis via the ERα-regulated ATF3/SLC7A11 axis, highlighting its therapeutic potential in SS.

POU2F2 activates the Akt/mTOR signalling pathway and enhances B lymphocyte function during diabetic kidney disease by promoting PIK3CD transcription.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) is the predominant isoform of the catalytic subunit of PI3K in lymphocytes. Based on comprehensive bioinformatics, this study explores the functions of PIK3CD in diabetic kidney disease (DKD) progression in mice and B lymphocyte activity. Non-obese diabetic (NOD) mice that spontaneously develop DKD were applied as animal models. Lentiviral vector-mediated PIK3CD silencing was introduced in mice, followed by histological staining and biomarker assessments to evaluate DKD-associated symptoms. The activity of the Akt/mTOR signalling pathway was determined by western blot analysis. Following bioinformatics analyses, the interaction between POU class 2 homeobox 2 (POU2F2) and PIK3CD in B lymphocytes was determined by chromatin immunoprecipitation and luciferase reporter assays. Their functions in B cell function were identified by MTT, flow cytometry and IgG deposition assessment. PIK3CD was found to be highly expressed in the kidney of DKD mice. Knockdown of PIK3CD inactivated the Akt/mTOR signalling, thus ameliorating tissue injury and fibrosis, enhancing the expression of AQP1 and decreasing urinary NGAL contents, UACR, BUN and β-NAG/creatinine ratio. These effects were negated by the Akt-specific activator SC79. POU2F2 was found to promote PIK3CD transcription by binding to its promoter, thus activating the Akt/mTOR pathway. Knockdown of POU2F2 suppressed B cell proliferation in vitro and decreased B cell population and IgG deposition in the mouse kidney. However, these trends were reversed upon additional PIK3CD overexpression. This study demonstrates that POU2F2 mediates PIK3CD-dependent Akt/mTOR signalling activation, thus enhancing B lymphocyte function and promoting DKD progression.

12527 Determining The Impact Of Dampened Oxidative Stress On Islet Function And Gene Expression During Diabetes Onset In NOD Mice

Abstract Disclosure: K.V. Coutinho: None. H. Shinde: None. J. Feduska: None. K. Burnette: None. S.O. Poole: None. H.M. Tse: None. C.S. Hunter: None. Type 1 diabetes (T1D) arises from autoimmune destruction of insulin-producing pancreatic β-cells. Revealing the mechanisms underlying T1D onset are critical for therapeutic development but not completely understood. NADPH oxidase (NOX) generates superoxide, a precursor of reactive oxygen species (ROS), to regulate cell survival, differentiation, signaling, and autoimmune responses in T1D. NOD (Non-Obese Diabetic) mice spontaneously develop T1D, featuring increased cytokine/chemokine synthesis, inflammatory responsive macrophages and T cells, and ROS. Here, we harness NOD mice deficient in NOX-derived superoxide (NOD.Ncf1m[1]J), which have significantly lower T1D incidence. However, the impacts on islet β-cells in this T1D resistant model are unknown. We assessed a time course of glucose homeostasis between 6-20 weeks (W), spanning the onset T1D in NOD mice. Surprisingly, comparative glucose tolerance testing (GTT) in NOD.Ncf1m[1]J mice revealed increased glucose intolerance at 10W with no differences at 6, 14, and 20W as compared to NOD. Insulin tolerance was similar between the two models at all stages examined. This suggests superoxide production is necessary for β-cell function, despite that NOD.Ncf1m[1]J mice are T1D resistant. To understand islet transcriptomic changes between NOD and NOD.Ncf1m[1]J mice, we conducted comparative bulk islet RNA sequencing from 6 to 20W. Principal component analysis was applied to ensure differences between genotypes at each timepoint. Quality control methods within the Nextflow core pipeline were used to ensure sample quality. As expected, NOD.Ncf1m[1]J mice had decreased Ncf1 and immune response-related mRNA levels. Expression of T-cell exhaustion (Pdcd1, Lag3, and Tigit) genes remained level in NOD mice, but had significantly lower expression at 6W in NOD.Ncf1m[1]J mice. Interestingly, Ctla4, encoding a gene product that inhibits T-cell function, was upregulated at 14W in NOD.Ncf1m[1]J mice. Furthermore, GO analysis of downregulated NOD.Ncf1m[1]J gene sets include T-cell activation, response to IFN-β and IFN-γ at 6W, and decreased cytokine production at 20W. Supporting the dysregulation of β-cell function, Bace2, encoding a protease that limits β-cell function and mass, was upregulated with NOD. Ncf1m[1]J. Additionally, 14W β-cell function gene expression was decreased in the NOD.Ncf1m[1]J mice, including Slc2a2, which encodes the β-cell GLUT2 glucose transporter, and Glp1r, which is involved in preserving β-cell mass and function. Taken together, NOX-derived superoxide, whose loss decreases T1D incidence, may also dampen β-cell gene expression and function. Future directions will include comparative NOD and NOD.Ncf1m[1]J single cell transcriptomics to determine cell type specific transcriptional signatures between islet and immune cell subtypes. Presentation: 6/2/2024

9343 Protection Against Type 1 Diabetes Development In Mice With 4E-BP2 Deletion

Abstract Disclosure: V. Pita Grisanti: None. F. Pecanha: None. R. Louzada: None. M. Blandino: None. C. Jaramillo: None. N. Arenas: None. A. Bayer: None. E. Bernal-Mizrachi: None. Type 1 diabetes is an autoimmune disease characterized by β-cell destruction promoted by autoreactive T cells that acquire an effector inflammatory phenotype. 4E-BP1/2 proteins are translational repressors and downstream targets of mTORC1, a key regulator of metabolism. mTORC1 signaling disruption is implicated in human diseases including diabetes. Activation of 4E-BP2/eIF4E pathway by 4E-BP2 deletion promotes translation initiation, which induces β-cell expansion and proliferation and is crucial for the regulation of adaptive immunity. However, the involvement of 4E-BP2 in type 1 diabetes development has not been explored. Therefore, this study aimed to determine the role of 4E-BP2/eIF4E signaling in type 1 diabetes prevention by regulation of β-cell survival and immune modulation. To investigate the role of 4E-BP2/eIF4E axis in diabetes pathogenesis, we used the non-obese diabetic (NOD) mouse model as a type 1 diabetes model, and generated mice with global deletion of 4E-BP2 in the NOD background (4E-BP2KONOD). We assessed type 1 diabetes development, glucose homeostasis, pancreas morphometry and immune responses in male and female 4E-BP2KONOD and control NOD mice. We found that 4E-BP2KONOD exhibited reduced diabetes incidence in male but not female mice. Reduction in diabetes incidence in 4E-BP2KONOD male mice was associated with comparable degree of insulitis and β-cell proliferation, and with preserved β-cell mass compared to the control NOD mice. Glucose-stimulated insulin secretion of the islets, assessed in vitro by static incubation, was significantly higher in the 4E-BP2KONOD compared to the NOD control. Autoimmune responses were also evaluated by assessment of insulitis and characterization of T cell compartments, which showed a decrease in splenic CD8+ cytotoxic T cells and an increase in pancreatic regulatory T cell (Treg) infiltration mainly by increased Treg survival in 4E-BP2KONOD mice compared to control NOD. Finally, adoptive transfer studies demonstrated that lymphocytes derived from male 4E-BP2KONOD mice were less diabetogenic than those derived from control NOD mice. In conclusion, this study showed that activation of 4E-BP2/eIF4E axis in 4E-BP2KONOD male mice protects against type 1 diabetes development by dampening autoimmune responses and preserving β-cell mass. Targeting 4E-BP2 may provide a potential treatment for type 1 diabetes. Presentation: 6/1/2024

8090 A Gut Commensal, Parabacteroides distasonis, and Molecular Mimicry in Type 1 Diabetes

Abstract Disclosure: A. Randall: None. Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic, insulin-producing beta cells by autoreactive T-cells. The trigger(s) of T1D are unclear and genetics alone cannot explain the recent increase in T1D incidence. The gut microbiome composition in T1D patients is altered and thought to play a key role in T1D pathogenesis. Most longitudinal studies have been descriptive. However, a direct cause-and-effect relationship hasn't been established yet. Insulin is a key autoantigen in T1D autoimmunity, and insulin B-chain amino acids 9-23 (insB:9-23) is an important, commonly recognized T-cell epitope. Molecular mimicry is an autoimmunity mechanism where host-like microbial proteins trigger autoimmune responses. We previously identified a peptide mimic of insB:9-23, amino acids 4-18 of hypoxanthine phosphoribosyl transferase (HPRT:4-18) of Parabacteroides distasonis (Girdhar et al., 2022). HPRT:4-18 stimulated insB:9-23-specific human and non-obese diabetic (NOD) mice T-cells. We hypothesize that P. distasonis HPRT:4-18 is required to accelerate diabetes via molecular mimicry of insB:9-23. By orally gavaging NOD mice (n=40 mice/group), we showed that P. distasonis accelerated diabetes onset. Next, we colonized NOD mice with another common gut commensal, Bacteroides fragilis, as a control, and did not observe any effect on islet infiltration. In addition, P. distasonis colonization increased infiltration of immune cells into the islets of germ-free NOD mice (n=6-8 mice/group), while the diabetes incidence experiment in mice is ongoing. P. distasonis-colonized mice had significantly more splenic CD8 and naïve T-cells, dendritic cells and macrophages but fewer FOXP3 cells (n=3-6 mice/group). To determine whether P. distasonis’s effect is specific to the pancreas, we checked intestinal barrier function and intestinal immune cell composition. Disruption of intestinal barrier function has been associated with diabetes. P. distasonis colonization did not alter gut permeability. Furthermore, P. distasonis did not increase intestinal inflammation. Among the intestinal intraepithelial lymphocytes (IELs), there were fewer naïve, central and effector T-cells and B-cells. The findings indicate that P. distasonis does not stimulate an unspecific immune response and its inflammatory effect is specific to the pancreas, supporting our initial hypothesis. Taken together, our results indicate that exposure to P. distasonis in the first years of life has the potential to stimulate molecular mimicry mechanism and cause T1D onset. Reference: Girdhar, K., Huang, Q., Chow, I. T., Vatanen, T., Brady, C., Raisingani, A., . . . Altindis, E. (2022). A gut microbial peptide and molecular mimicry in the pathogenesis of type 1 diabetes. Proc Natl Acad Sci U S A, 119(31), e2120028119. doi:10.1073/pnas.2120028119 Presentation: 6/2/2024

8439 Targeting Interferon Gamma-Mediated CXCL16-CXCR6 Chemokine-Receptor Interactions As A Strategy To Prevent Immune Checkpoint Inhibitor Autoimmune Diabetes

Abstract Disclosure: S. Wang: None. N. Huang: None. J.G. Ortega: None. J. Lee: None. K. Kimbrell: None. J. Nguyen: None. J. Olay: None. E. McCarthy: None. E. Peluso: None. H. Chen: None. M.G. Lechner: None. Autoimmune diabetes mellitus is a rare but life-threatening side effect of immune checkpoint inhibitor (ICI) cancer therapy. Checkpoint inhibitors increase immune activation by blocking regulatory molecules on T cells, including programmed death protein (PD1). As a result of this heightened immune activation, patients can develop autoimmunity in healthy tissues, known as immune related adverse events (IRAEs). ICI-associated diabetes mellitus (ICI-T1DM) is an IRAE that results in autoimmune destruction of insulin-producing pancreatic beta-cells. Patients with ICI-T1DM require life-long insulin therapy and more than half present initially with diabetic ketoacidosis. Unfortunately, the mechanism of this IRAE is not fully understood. Anti-PD1 antibody treatment (10mg/kg/dose, twice weekly, i.p.) of male and female non-obese diabetic (NOD) mice leads to autoimmune infiltrates in multiple tissues, including accelerated insulitis and DM. Single cell RNA sequencing of islet infiltrating immune cells from ICI-treated mice previously showed a role for CD8+ T cells and cytokine interferon gamma (IFNγ) in the development of ICI-T1DM. Using CellChat to perform receptor-ligand interaction analysis of these data, we identified both CD8+ and CD4+ T cells as the primary source of IFNγ in immune infiltrates and islet myeloid cells as the primary target. Furthermore, CellChat analysis showed increased receptor-ligand interactions between T cell receptor CXCR6 and its chemokine CXCL16, as well as CXCR3 and chemokines CXCL9 and CXCL10. In vitro IFNγ stimulation of myeloid cells induced strong expression of CXCL16, 9, and 10, suggesting a potential mechanism by which effector T cells are recruited to islets during ICI therapy. Immunophenotyping of islet-infiltrating immune cells indeed showed increased IFNγ+CXCR6+ effector CD8+ cells in anti-PD1 treated mice compared to isotype-treated controls. Moreover, these IFNγ+CXCR6+CD8+ T cells stained positively for the NRP-V7 mimotope tetramer, a known antigen for CD8+ T cells in spontaneous autoimmune diabetes in NOD mice, suggesting this as an antigen-specific population contributing to the immunopathogenesis of ICI-T1DM. Finally, genetic deletion of CXCR6 in NOD mice delayed the onset of autoimmune diabetes during anti-PD1 treatment in female (p=0.0162) but not male mice (p=0.3656). In summary, these data further elucidate the cellular mechanisms by which IFNγ and chemokine signaling pathways drive the development of ICI-T1DM and identify the CXCR6-CXCL16 axis as a potential therapeutic target to prevent this IRAE in checkpoint inhibitor-treated cancer patients. Presentation: 6/2/2024

Mammalian D-Cysteine controls insulin secretion in the pancreas

D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Prevention of Diabetic Nephropathy with Mesenchymal Stromal Cells in Cardiac Surgery Patients and in Nonobese Diabetic (NOD) Mice and Diabetic Dogs with "Neo-Islets," Three-Dimensional Organoids of Mesenchymal Stem Cells (MSCs), and Pancreatic Islet Cells

Prevention of Diabetic Nephropathy with Mesenchymal Stromal Cells in Cardiac Surgery Patients and in Nonobese Diabetic (NOD) Mice and Diabetic Dogs with "Neo-Islets," Three-Dimensional Organoids of Mesenchymal Stem Cells (MSCs), and Pancreatic Islet Cells

The impact of alternate-day fasting on the salivary gland stem cell compartments in non-obese diabetic mice with newly established Sjögren's syndrome.

Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAMhi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16INK4a, a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS.

Targeting the autoreactive CD8+ T-cell receptor in type 1 diabetes: Insights from scRNA-seq for immunotherapy

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the attack and destruction of Pancreatic islet beta cells by T cells. Understanding the role of T-cell receptor (TCR) in the development of T1D is of paramount importance. This study employs single-cell RNA sequencing (scRNA-seq) to delve into the mechanistic actions and potential therapeutic applications of autoreactive stem cell-like CD8 TCR in T1D. By retrieving T-cell data from non-obese diabetic (NOD) mice via the GEO database, it was revealed that CD8+ T cells are the predominant T-cell subset in the pancreatic tissue of T1D mice, along with the identification of T-cell marker genes closely associated with T1D. Moreover, the gene TRAJ23 exhibits a preference for T1D, and its knockout alleviates T1D symptoms and adverse reactions in NOD mice. Additionally, engineered TCR-T cells demonstrate significant cytotoxicity towards β cells in T1D.

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes.

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. 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. 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. 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.

Differential lipid signaling from CD4+ and CD8+ T cells contributes to type 1 diabetes development.

We reported that Ca2+-independent phospholipase A2β (iPLA2β)-derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting β-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development. CD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2β+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid. In mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%-50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive β-cells. Reduced iPLA2β led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B. These findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.

Regulatory CD4+ T cells redirected against pathogenic CD8+ T cells protect NOD mice from development of autoimmune diabetes.

In this study, we report a novel therapeutic approach redirecting antigen-specific CD4+ T cells recognizing a hybrid insulin peptide (BDC2.5 T cell receptor (TCR) transgenic CD4+ T cells) to attract and suppress islet-specific CD8+ T cells T cells in the non-obese diabetic (NOD) mouse model, and prevent the development of autoimmune diabetes. Purified BDC2.5 CD4+ T cells were induced to differentiate into regulatory T cells (Tregs). The Tregs were then electroporated with mRNA encoding chimeric human β2 microglobulin (hβ2m) covalently linked to insulin B chain amino acids 15-23 (designated INS-eTreg) or islet-specific glucose-6-phosphatase related protein (IGRP) peptide 206-214 (designated IGRP-eTreg). Immunoregulatory functions of these engineered regulatory T cells (eTregs) were tested by in vitro assays and in vivo co-transfer experiments with β-cell-antigen-specific CD8+ T cells in NOD.Scid mice or by adoptive transfer into young, pre-diabetic NOD mice. These eTregs were phenotyped by flow cytometry, and shown to have high expression of FoxP3, as well as other markers of Treg function, including IL-10. They suppressed polyclonal CD4+ T cells and antigen-specific CD8+ T cells (recognizing insulin or IGRP), decreasing proliferation and increasing exhaustion and regulatory markers in vitro. In vivo, eTregs reduced diabetes development in co-transfer experiments with pathogenic antigen-specific CD8+ T cells (INS-CD8+ or IGRP-CD8+ cells) into NOD.Scid mice. Finally, when the eTreg were injected into young NOD mice, they reduced insulitis and prevented spontaneous diabetes in the recipient mice. Our results suggest a novel therapeutic strategy to protect NOD mice by targeting antigen-specific cytotoxic CD8+ T cells, using redirected antigen-specific CD4+ Treg cells, to suppress autoimmune diabetes. This may suggest an innovative therapy for protection of people at risk of development of type 1 diabetes.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

Selective Reduction of Ca2+-Independent Phospholipase A2β (iPLA2β)-Derived Lipid Signaling from Macrophages Mitigates Type 1 Diabetes Development.

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of β-cells and macrophages (MΦ) have a central role in initiating processes that lead to β-cell demise. We reported that Ca2+-independent phospholipase A2β (iPLA2β)-derived lipid (iDL) signaling contributes to β-cell death. As MΦ express iPLA2β, we assessed its role in T1D development. We find that selective reduction of myeloid-iPLA2β in spontaneously diabetes-prone nonobese diabetic (NOD) mice (a) deceases proinflammatory eicosanoid production by MΦ, (b) favors anti-inflammatory (M2-like) MΦ phenotype, and (c) diminishes activated CD4+ and CD8+ T-cells phenotype in the pancreatic infiltrate, prior to T1D onset. These outcomes are associated with a significant reduction in T1D. Further, inhibition of select proinflammatory lipid signaling pathways reduces M1-like MΦ polarization and adoptive transfer of M2-like MΦ reduces NOD T1D incidence, suggesting a mechanism by which iDLs impact T1D development. These findings identify MΦ-iPLA2β as a critical contributor to TID development and potential target to counter T1D onset.

The Impact of Light-Dark Cycle Alteration on the Acceleration of Type 1 Diabetes in NOD Mice Model.

We aimed to evaluate the effect of light-dark cycle alteration and soft drink consumption on the acceleration of type 1 diabetes mellitus (T1DM) development among non-obese diabetic (NOD) mice model. We exposed female NOD and C57BL/6 mice from the age of 5 weeks to either adlib soft drink consumption and/or T20 light-dark cycle alteration until the development of diabetes, or the mice reached the age of 30 weeks. Each group consisted of 7-15 mice. We monitored weight, length, blood glucose level, and insulin autoantibody (IAA) levels weekly. Out of 75 NOD and 22 C57BL/6 mice, 41 NOD mice developed diabetes, and 6 mice died between 7 and 8 weeks of age. The mean time to development of T1DM among NOD control mice was 20 weeks. The time to development of T1DM was accelerated by two weeks in the NOD mice exposed to light-dark cycle alteration, hazard ratio of 2.65,95th CI (0.70, 10.04) p = 0.15). The other groups developed T1DM, similar to the control group. There was a trend toward earlier development of T1DM among NOD mice exposed to light-dark cycle alteration, but this difference was not statistically significant. Further studies are needed to confirm our findings using larger sample sizes and different animal species.

Maidong Dishao Decoction mitigates submandibular gland injury in NOD mice through modulation of gut microbiota and restoration of Th17/Treg immune balance

BackgroundPrimary Sjogren's syndrome (pSS) is a common chronic autoimmune disease that presents limited treatment options and poses significant challenges for patients. Maidong Dishao Decoction (MDDST), a traditional Chinese medicine compound, has demonstrated potential in alleviating dryness symptoms associated with pSS. Therefore, it is important to study the specific mechanism of its therapeutic effect. ObjectiveThis study aims to investigate the effects of MDDST on gut microbiota, short-chain fatty acids (SCFAs), and the Th17/Treg immune balance in non-obese diabetes (NOD) mice. MethodsThe study employed ultrahigh-performance liquid chromatography coupled with quadrupole-exactive mass spectrometry (UHPLC-QE-MS) to identify the primary components of MDDST. Subsequently, hematoxylin and eosin (HE) staining, enzyme-linked immunosorbent assays (ELISA), and flow cytometry analyses were conducted to evaluate the therapeutic effects of MDDST in NOD mice. Additionally, 16S rDNA sequencing and gas chromatography-mass spectrometry (GC-MS) were utilized to assess the influence of MDDST on gut microbiota and SCFAs. Finally, fecal microbiota transplantation (FMT) and SCFA-based interventions were performed to elucidate the mechanisms through which MDDST exerts its effects. ResultsThe research findings demonstrate that MDDST exerts therapeutic effects on NOD mice, primarily manifested as reduced inflammation, decreased water intake, ameliorated pathological changes and lowered levels of Sjogren's syndrome antigen A (SSA) and immunoglobulin G (IgG). Additionally, MDDST significantly decreased serum levels of interleukin-6 (IL-6) and interleukin-17 (IL-17), while enhancing levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), thereby regulating the Th17/Treg immune balance. Further investigations revealed that MDDST treatment induces alterations in gut microbiota composition and elevates SCFA levels in the gut. Subsequent FMT and SCFA intervention experiments demonstrated a downregulation of pSS-related phenotypes. ConclusionIn summary, MDDST demonstrates protective effects against pSS by restoring the balance between Th17 and Treg cells. The therapeutic effects can be partially attributed to its regulation of gut microbiota and SCFAs. Our finding provides a new option for treating pSS.

Mitochondria-targeted NIR molecular probe for detecting viscosity of gland damage and SO2 in actual samples

Glandular damage can be caused by various factors, including disease, trauma, or other abnormalities within the organism. The viscosity of the gland is one of the important indicators to measure the degree of damage. Sulfur dioxide (SO2) is widely used as an important food additive due to its preservative and bleaching properties, but its overuse has serious negative impacts on the environment, so it is urgent to develop a simple detection method. Herein, we designed and synthesized a mitochondria-targeted near-infrared (NIR) fluorescence probe (BDC) for the detection of viscosity and SO2. BDC consisted of a donor-π-acceptor (D-π-A) structure and extended double bonds bridging rotor, which enabled sensitive response to viscosity and intense fluorescence emission. The TICT (twisted intramolecular charge transfer) of BDC was inhibited with an increase in viscosity, accompanied by a significant enhancement of red fluorescence signal with emission wavelength beyond 800 nm. Notably, BDC was able to noninvasively and sensitively monitor the viscosity changes in the glands of non-obese diabetic (NOD) mice model. BDC was utilized for monitoring SO2 in food and environmental samples through Michael addition reactions, providing a straightforward tool for SO2 detection in food safety and environmental monitoring.

CD4+ T cells reactive to a hybrid peptide from insulin-chromogranin A adopt a distinct effector fate and are pathogenic in autoimmune diabetes

T cell-mediated islet destruction is a hallmark of autoimmune diabetes. Here, we examined the dynamics and pathogenicity of CD4+ Tcell responses to four different insulin-derived epitopes during diabetes initiation in non-obese diabetic (NOD) mice. Single-cell RNA sequencing of tetramer-sorted CD4+ Tcells from the pancreas revealed that islet-antigen-specific Tcells adopted a wide variety of fates and required XCR1+ dendritic cells for their activation. Hybrid-insulin C-chromogranin A (InsC-ChgA)-specific CD4+ Tcells skewed toward a distinct T helper type 1 (Th1) effector phenotype, whereas the majority of insulin B chain and hybrid-insulin C-islet amyloid polypeptide-specific CD4+ T cells exhibited a regulatory phenotype and early or weak Th1 phenotype, respectively. InsC-ChgA-specific CD4+ Tcells were uniquely pathogenic upon transfer, and an anti-InsC-ChgA:IAg7 antibody prevented spontaneous diabetes. Our findings highlight the heterogeneity of Tcell responses to insulin-derived epitopes in diabetes and argue for the feasibility of antigen-specific therapies that blunts the response of pathogenic CD4+ Tcells causing autoimmunity.