The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Transcriptomic Differences in Peripheral Monocyte Populations in Septic Patients Based on Outcome.

ABSTRACT: Post-sepsis early mortality is being replaced by survivors who experience either a rapid recovery and favorable hospital discharge or the development of chronic critical illness (CCI) with suboptimal outcomes. The underlying immunological response that determines these clinical trajectories remains poorly defined at the transcriptomic level. As classical and non-classical monocytes are key leukocytes in both the innate and adaptive immune systems, we sought to delineate the transcriptomic response of these cell types. Using single-cell RNA sequencing and pathway analyses, we identified gene expression patterns between these two groups that are consistent with differences in TNFα production based on clinical outcome. This may provide therapeutic targets for those at risk for CCI in order to improve their phenotype/endotype, morbidity, and long-term mortality.

Serum from pregnant donors induces human beta cell proliferation.

Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-ßH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-ßH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.

Proteome-scale tissue mapping using mass spectrometry based on label-free and multiplexed workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in both normal and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput. Since many of these existing challenges are associated with mass spectrometry-based protein identification and quantification, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ~3500 proteins at a spatial resolution of 50 µm and the largest quantification dynamic range, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provide robust protein quantifications in terms of identifying differentially abundant proteins and spatially co-variable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables to identify protein markers specific to different cell types, but more importantly, it also reveals unknown or hidden protein patterns by spatial co-expression analysis.

Redox regulation of m6A methyltransferase METTL3 in ß-cells controls the innate immune response in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by the destruction of pancreatic ß-cells. Several observations have renewed the interest in ß-cell RNA sensors and editors. Here, we report that N6-methyladenosine (m6A) is an adaptive ß-cell safeguard mechanism that controls the amplitude and duration of the antiviral innate immune response at T1D onset. m6A writer methyltransferase 3 (METTL3) levels increase drastically in ß-cells at T1D onset but rapidly decline with disease progression. m6A sequencing revealed the m6A hypermethylation of several key innate immune mediators, including OAS1, OAS2, OAS3 and ADAR1 in human islets and EndoC-ßH1 cells at T1D onset. METTL3 silencing enhanced 2'-5'-oligoadenylate synthetase levels by increasing its mRNA stability. Consistently, in vivo gene therapy to prolong Mettl3 overexpression specifically in ß-cells delayed diabetes progression in the non-obese diabetic mouse model of T1D. Mechanistically, the accumulation of reactive oxygen species blocked upregulation of METTL3 in response to cytokines, while physiological levels of nitric oxide enhanced METTL3 levels and activity. Furthermore, we report that the cysteines in position C276 and C326 in the zinc finger domains of the METTL3 protein are sensitive to S-nitrosylation and are important to the METTL3-mediated regulation of oligoadenylate synthase mRNA stability in human ß-cells. Collectively, we report that m6A regulates the innate immune response at the ß-cell level during the onset of T1D in humans.

NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

Low-Dose Antithymocyte Globulin: A Pragmatic Approach to Treating Stage 2 Type 1 Diabetes.

OBJECTIVE: Low-dose antithymocyte globulin (ATG) (2.5 mg/kg) preserves C-peptide and reduces HbA1c in new-onset stage 3 type 1 diabetes, yet efficacy in delaying progression from stage 2 to stage 3 has not been evaluated. RESEARCH DESIGN AND METHODS: Children (n = 6) aged 5-14 years with stage 2 type 1 diabetes received off-label, low-dose ATG. HbA1c, C-peptide, continuous glucose monitoring, insulin requirements, and side effects were followed for 18-48 months. RESULTS: Three subjects (50%) remained diabetes free after 1.5, 3, and 4 years of follow-up, while three developed stage 3 within 1-2 months after therapy. Eighteen months posttreatment, even disease progressors demonstrated near-normal HbA1c (5.1% [32 mmol/mol], 5.6% [38 mmol/mol], and 5.3% [34 mmol/mol]), time in range (93%, 88%, and 98%), low insulin requirements (0.17, 0.18, and 0.34 units/kg/day), and robust C-peptide 90 min after mixed meal (1.3 ng/dL, 2.3 ng/dL, and 1.4 ng/dL). CONCLUSIONS: These observations support additional prospective studies evaluating ATG in stage 2 type 1 diabetes.

Deletion of Vß3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vß3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vß3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vß3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vß repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

Alpha cell receptor for advanced glycation end products associate with glucagon expression in type 1 diabetes.

Hypoglycemia in type 1 diabetes associates with changes in the pancreatic islet α cells, where the receptor for advanced glycation end products (RAGE) is highly expressed. This study compared islet RAGE expression in donors without diabetes, those at risk of, and those with type 1 diabetes. Laser-dissected islets were subject to RNA bioinformatics and adjacent pancreatic tissue were assessed by confocal microscopy. We found that islets from type 1 diabetes donors had differential expression of the RAGE gene (AGER) and its correlated genes, based on glucagon expression. Random forest machine learning revealed that AGER was the most important predictor for islet glucagon levels. Conversely, a generalized linear model identified that glucagon expression could be predicted by expression of RAGE signaling molecules, its ligands and enzymes that create or clear RAGE ligands. Confocal imaging co-localized RAGE, its ligands and signaling molecules to the α cells. Half of the type 1 diabetes cohort comprised of adolescents and a patient with history of hypoglycemia-all showed an inverse relationship between glucagon and RAGE. These data confirm an association between glucagon and islet RAGE, its ligands and signaling pathways in type 1 diabetes, which warrants functional investigation into a role for RAGE in hypoglycemia.

Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes.

BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

Human immune phenotyping reveals accelerated aging in type 1 diabetes.

The proportions and phenotypes of immune cell subsets in peripheral blood undergo continual and dramatic remodeling throughout the human life span, which complicates efforts to identify disease-associated immune signatures in type 1 diabetes (T1D). We conducted cross-sectional flow cytometric immune profiling on peripheral blood from 826 individuals (stage 3 T1D, their first-degree relatives, those with ≥2 islet autoantibodies, and autoantibody-negative unaffected controls). We constructed an immune age predictive model in unaffected participants and observed accelerated immune aging in T1D. We used generalized additive models for location, shape, and scale to obtain age-corrected data for flow cytometry and complete blood count readouts, which can be visualized in our interactive portal (ImmScape); 46 parameters were significantly associated with age only, 25 with T1D only, and 23 with both age and T1D. Phenotypes associated with accelerated immunological aging in T1D included increased CXCR3+ and programmed cell death 1-positive (PD-1+) frequencies in naive and memory T cell subsets, despite reduced PD-1 expression levels on memory T cells. Phenotypes associated with T1D after age correction were predictive of T1D status. Our findings demonstrate advanced immune aging in T1D and highlight disease-associated phenotypes for biomarker monitoring and therapeutic interventions.

Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells.

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing ß-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human ß-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes.

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.

Serum from pregnant donors induces human beta cell proliferation and insulin secretion.

Pancreatic beta cells are among the slowest replicating cells in the human body. Human beta cells usually do not increase in number with exceptions being during the neonatal period, in cases of obesity, and during pregnancy. This project explored maternal serum for stimulatory potential on human beta cell proliferation and insulin output. Gravid, full-term women who were scheduled to undergo cesarean delivery were recruited for this study. A human beta cell line was cultured in media supplemented with serum from pregnant and non-pregnant donors and assessed for differences in proliferation and insulin secretion. A subset of pregnant donor sera induced significant increases in beta cell proliferation and insulin secretion. Pooled serum from pregnant donors also increased proliferation in primary human beta cells but not primary human hepatocytes indicating a cell-type specific effect. This study suggests stimulatory factors in human serum during pregnancy could provide a novel approach for human beta cell expansion.

Redox Regulation of m 6 A Methyltransferase METTL3 in Human ß-cells Controls the Innate Immune Response in Type 1 Diabetes.

Type 1 Diabetes (T1D) is characterized by autoimmune-mediated destruction of insulin-producing ß-cells. Several observations have renewed interest in the innate immune system as an initiator of the disease process against ß-cells. Here, we show that N 6 -Methyladenosine (m 6 A) is an adaptive ß-cell safeguard mechanism that accelerates mRNA decay of the 2'-5'-oligoadenylate synthetase (OAS) genes to control the antiviral innate immune response at T1D onset. m 6 A writer methyltransferase 3 (METTL3) levels increase drastically in human and mouse ß-cells at T1D onset but rapidly decline with disease progression. Treatment of human islets and EndoC-ßH1 cells with pro-inflammatory cytokines interleukin-1 ß and interferon α mimicked the METTL3 upregulation seen at T1D onset. Furthermore, m 6 A-sequencing revealed the m 6 A hypermethylation of several key innate immune mediators including OAS1, OAS2, and OAS3 in human islets and EndoC-ßH1 cells challenged with cytokines. METTL3 silencing in human pseudoislets or EndoC-ßH1 cells enhanced OAS levels by increasing its mRNA stability upon cytokine challenge. Consistently, in vivo gene therapy, to prolong Mettl3 overexpression specifically in ß-cells, delayed diabetes progression in the non-obese diabetic (NOD) mouse model of T1D by limiting the upregulation of Oas pointing to potential therapeutic relevance. Mechanistically, the accumulation of reactive oxygen species blocked METTL3 upregulation in response to cytokines, while physiological levels of nitric oxide promoted its expression in human islets. Furthermore, for the first time to our knowledge, we show that the cysteines in position C276 and C326 in the zinc finger domain of the METTL3 protein are sensitive to S-nitrosylation (SNO) and are significant for the METTL3 mediated regulation of OAS mRNA stability in human ß-cells in response to cytokines. Collectively, we report that m 6 A regulates human and mouse ß-cells to control the innate immune response during the onset of T1D and propose targeting METTL3 to prevent ß-cell death in T1D.

Intergenomic and epistatic interactions control free radical mediated pancreatic ß-cell damage.

Alloxan (AL)-generated Reactive Oxygen Species (ROS) selectively destroy insulin-producing pancreatic ß-cells. A previous genome-wide scan (GWS) using a cohort of 296 F2 hybrids between NOD (AL-sensitive) and ALR (AL-resistant) mice identified linkages contributing to ß-cell susceptibility or resistance to AL-induced diabetes on Chromosomes (Chr) 2, 3, 8, and a single nucleotide polymorphism in mt-Nd2 of the mitochondrial genome (mtDNA). AL treatment of congenic and consomic NOD mouse stocks confirmed resistance linked to both the mtDNA and the Chr 8 locus from ALR [NOD.mtALR.ALR-(D8Mit293-D8Mit137)]. To identify possible epistatic interactions, the GWS analysis was expanded to 678 F2 mice. ALR-derived diabetes-resistance linkages on Chr 8 as well as the mt-Nd2 a allele were confirmed and novel additional linkages on Chr 4, 5, 6, 7, and 13 were identified. Epistasis was observed between the linkages on Chr 8 and 2 and Chr 8 and 6. Furthermore, the mt-Nd2 genotype affected the epistatic interactions between Chr 8 and 2. These results demonstrate that a combination of nuclear-cytoplasmic genome interactions regulates ß-cell sensitivity to ROS-mediated ALD.

Detection of Antiviral Tissue Responses and Increased Cell Stress in the Pancreatic Islets of Newly Diagnosed Type 1 Diabetes Patients: Results From the DiViD Study.

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls. Methods: RNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue. Results: The data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of ß-cells displayed markedly increased PKR protein levels. These PKR+ ß-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain ß-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in ß-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes ß-cells. Conclusion/interpretation: The gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in ß-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet ß-cells contributes to the pathogenesis of type 1 diabetes.

Adaptation to chronic ER stress enforces pancreatic ß-cell plasticity.

Pancreatic ß-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise ß-cell identity is unknown. We show here under reversible, chronic stress conditions ß-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of ß-cell function and identity. Upon recovery from stress, ß-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while ß-cells show resilience to episodic ER stress, when episodes exceed a threshold, ß-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest ß-cell adaptive exhaustion contributes to diabetes pathogenesis.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

The Foundation for Engineering a Pancreatic Islet Niche.

Progress in diabetes research is hindered, in part, by deficiencies in current experimental systems to accurately model human pathophysiology and/or predict clinical outcomes. Engineering human-centric platforms that more closely mimic in vivo physiology, however, requires thoughtful and informed design. Summarizing our contemporary understanding of the unique and critical features of the pancreatic islet can inform engineering design criteria. Furthermore, a broad understanding of conventional experimental practices and their current advantages and limitations ensures that new models address key gaps. Improving beyond traditional cell culture, emerging platforms are combining diabetes-relevant cells within three-dimensional niches containing dynamic matrices and controlled fluidic flow. While highly promising, islet-on-a-chip prototypes must evolve their utility, adaptability, and adoptability to ensure broad and reproducible use. Here we propose a roadmap for engineers to craft biorelevant and accessible diabetes models. Concurrently, we seek to inspire biologists to leverage such tools to ask complex and nuanced questions. The progenies of such diabetes models should ultimately enable investigators to translate ambitious research expeditions from benchtop to the clinic.