A static glucose-stimulated insulin secretion (sGSIS) assay that is significantly predictive of time to diabetes reversal in the human islet bioassay.

INTRODUCTION: Static incubation (static glucose-stimulated insulin secretion, sGSIS) is a measure of islet secretory function. The Stimulation Index (SI; insulin produced in high glucose/insulin produced in low glucose) is currently used as a product release criterion of islet transplant potency. RESEARCH DESIGN AND METHODS: Our hypothesis was that the Delta, insulin secreted in high glucose minus insulin secreted in low glucose, would be more predictive. To evaluate this hypothesis, sGSIS was performed on 32 consecutive human islet preparations, immobilizing the islets in a slurry of Sepharose beads to minimize mechanical perturbation. Simultaneous full-mass subrenal capsular transplants were performed in chemically induced diabetic immunodeficient mice. Logistic regression analysis was used to determine optimal cut-points for diabetes reversal time and the Fisher Exact Test was used to assess the ability of the Delta and the SI to accurately classify transplant outcomes. Receiver operating characteristic curve analysis was performed on cut-point grouped data, assessing the predictive power and optimal cut-point for each sGSIS potency metric. Finally, standard Kaplan-Meier-type survival analysis was conducted. RESULTS: In the case of the sGSIS the Delta provided a superior islet potency metric relative to the SI.ConclusionsThe sGSIS Delta value is predicitive of time to diabetes reversal in the full mass human islet transplant bioassay.

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.

GABA and Combined GABA with GAD65-Alum Treatment Alters Th1 Cytokine Responses of PBMCs from Children with Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease culminating in the destruction of insulin-producing pancreatic cells. There is a need for the development of novel antigen-specific strategies to delay cell destruction, including combinatorial strategies that do not elicit systemic immunosuppression. Gamma-aminobutyric acid (GABA) is expressed by immune cells, ß-cells, and gut bacteria and is immunomodulatory. Glutamic-acid decarboxylase 65 (GAD65), which catalyzes GABA from glutamate, is a T1D autoantigen. To test the efficacy of combinatorial GABA treatment with or without GAD65-immunization to dampen autoimmune responses, we enrolled recent-onset children with T1D in a one-year clinical trial (ClinicalTrials.gov NCT02002130) and examined T cell responses. We isolated peripheral blood mononuclear cells and evaluated cytokine responses following polyclonal activation and GAD65 rechallenge. Both GABA alone and GABA/GAD65-alum treatment inhibited Th1 cytokine responses over the 12-month study with both polyclonal and GAD65 restimulation. We also investigated whether patients with HLA-DR3-DQ2 and HLA-DR4-DQ8, the two highest-risk human leukocyte antigen (HLA) haplotypes in T1D, exhibited differences in response to GABA alone and GABA/GAD65-alum. HLA-DR4-DQ8 patients possessed a Th1-skewed response compared to HLA-DR3-DQ2 patients. We show that GABA and GABA/GAD65-alum present an attractive immunomodulatory treatment for children with T1D and that HLA haplotypes should be considered.

Pancreatic islet cell type-specific transcriptomic changes during pregnancy and postpartum.

Pancreatic ß-cell mass expands during pregnancy and regresses in the postpartum period in conjunction with dynamic metabolic demands on maternal glucose homeostasis. To understand transcriptional changes driving these adaptations in ß-cells and other islet cell types, we performed single-cell RNA sequencing on islets from virgin, late gestation, and early postpartum mice. We identified transcriptional signatures unique to gestation and the postpartum in ß-cells, including induction of the AP-1 transcription factor subunits and other genes involved in the immediate-early response (IEGs). In addition, we found pregnancy and postpartum-induced changes differed within each endocrine cell type, and in endothelial cells and antigen-presenting cells within islets. Together, our data reveal insights into cell type-specific transcriptional changes responsible for adaptations by islet cells to pregnancy and their resolution postpartum.

Nanothin Conformal Coating with Poly(N-vinylpyrrolidone) and Tannic Acid (PVPON/TA) Preserves Murine and Human Pancreatic Islets Function.

Beta cell replacement therapies can restore glycemic control to select individuals living with type 1 diabetes. However, the obligation of lifelong immunosuppression restricts cell therapies from replacing exogenous insulin administration. Encapsulation strategies can reduce the inherent adaptive immune response; however, few are successfully translated into clinical testing. Herein, we evaluated if the conformal coating of islets with poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) (PVPON/TA) could preserve murine and human islet function while conferring islet allograft protection. In vitro function was evaluated using static glucose-stimulated insulin secretion, oxygen consumption rates, and islet membrane integrity. In vivo function was evaluated by transplanting human islets into diabetic immunodeficient B6.129S7-Rag1tm1Mom/J (Rag-/-) mice. The immunoprotective capacity of the PVPON/TA-coating was assessed by transplanting BALB/c islets into diabetic C57BL/6 mice. Graft function was evaluated by non-fasting blood glucose measurements and glucose tolerance testing. Both coated and non-coated murine and human islets exhibited indistinguishable in vitro potency. PVPON/TA-coated and control human islets were able to restore euglycemia post-transplant. The PVPON/TA-coating as monotherapy and adjuvant to systemic immunosuppression reduced intragraft inflammation and delayed murine allograft rejection. This study demonstrates that PVPON/TA-coated islets may be clinically relevant as they retain their in vitro and in vivo function while modulating post-transplant immune responses.

Localized cytotoxic T cell-associated antigen 4 and antioxidant islet encapsulation alters macrophage signaling and induces regulatory and anergic T cells to enhance allograft survival.

The loss of functional ß-cell mass is a hallmark of type 1 diabetes. Islet transplantation represents a promising alternative approach, but immune-mediated graft destruction remains a major challenge. We sought to use islet encapsulation technologies to improve graft survival and function without systemic immunosuppression. We hypothesized islet encapsulation with nanothin coatings consisting of tannic acid (TA), an antioxidant; poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer; and cytotoxic T cell-associated antigen 4 immunoglobulin (CTLA-4-Ig), an inhibitory immune receptor, will elicit localized immunosuppression to prolong islet allograft function and suppress effector T cell responses. In the absence of systemic immunosuppression, we demonstrated (PVPON/TA/CTLA-4-Ig)-encapsulated NOD.Rag islet grafts maintain function significantly longer than control IgG-containing (PVPON/TA/IgG) and nonencapsulated controls after transplantation into diabetic C57BL/6 mice. This protection coincided with diminished proinflammatory macrophage responses mediated by signal transducer and activator of transcription 1 signaling, decreased proinflammatory T cell effector responses, and CTLA-4-Ig-specific concomitant increases in anergic CD4+ T cells and regulatory T cells. Our results provide evidence that conjugation of CTLA-4-Ig to (PVPON/TA) coatings can suppress T cell activation, enhance regulatory T cell populations, prolong islet allograft survival, and induce localized immunosuppression after transplantation.

MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance.

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic ß cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

A randomized trial of oral gamma aminobutyric acid (GABA) or the combination of GABA with glutamic acid decarboxylase (GAD) on pancreatic islet endocrine function in children with newly diagnosed type 1 diabetes.

Gamma aminobutyric acid(GABA) is synthesized by glutamate decarboxylase(GAD) in ß-cells. Regarding Type 1 diabetes(T1D), animal/islet-cell studies found that GABA promotes insulin secretion, inhibits α-cell glucagon and dampens immune inflammation, while GAD immunization may also preserve ß-cells. We evaluated the safety and efficacy of oral GABA alone, or combination GABA with GAD, on the preservation of residual insulin secretion in recent-onset T1D. Herein we report a single-center, double-blind, one-year, randomized trial in 97 children conducted March 2015 to June 2019(NCT02002130). Using a 2:1 treatment:placebo ratio, interventions included oral GABA twice-daily(n = 41), or oral GABA plus two-doses GAD-alum(n = 25), versus placebo(n = 31). The primary outcome, preservation of fasting/meal-stimulated c-peptide, was not attained. Of the secondary outcomes, the combination GABA/GAD reduced fasting and meal-stimulated serum glucagon, while the safety/tolerability of GABA was confirmed. There were no clinically significant differences in glycemic control or diabetes antibody titers. Given the low GABA dose for this pediatric trial, future investigations using higher-dose or long-acting GABA formulations, either alone or with GAD-alum, could be considered, although GABA alone or in combination with GAD-alum did nor preserve beta-cell function in this trial.

Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes.

Hybrid insulin peptides (HIPs) form in pancreatic ß-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with type 1 diabetes (T1D) as well as by pathogenic CD4 T-cell clones in nonobese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow the autoimmune destruction of ß-cells mediated by HIP-reactive CD4 T cells in T1D.

Carbonyl Posttranslational Modification Associated With Early-Onset Type 1 Diabetes Autoimmunity.

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various posttranslational modifications to self-proteins. In this study, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in NOD mice. Of interest, we identified carbonyl modification of the prolyl-4-hydroxylase ß subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found that carbonylated P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin-to-insulin ratios. Autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Moreover, we identify autoreactive CD4+ T-cell responses toward carbonyl-P4Hb epitopes in the circulation of patients with type 1 diabetes. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and in creating autoantigenic forms of insulin in type 1 diabetes.

Exploratory study reveals far reaching systemic and cellular effects of verapamil treatment in subjects with type 1 diabetes.

Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

The role of NADPH oxidases in infectious and inflammatory diseases.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) are enzymes that generate superoxide or hydrogen peroxide from molecular oxygen utilizing NADPH as an electron donor. There are seven enzymes in the NOX family: NOX1-5 and dual oxidase (DUOX) 1-2. NOX enzymes in humans play important roles in diverse biological functions and vary in expression from tissue to tissue. Importantly, NOX2 is involved in regulating many aspects of innate and adaptive immunity, including regulation of type I interferons, the inflammasome, phagocytosis, antigen processing and presentation, and cell signaling. DUOX1 and DUOX2 play important roles in innate immune defenses at epithelial barriers. This review discusses the role of NOX enzymes in normal physiological processes as well as in disease. NOX enzymes are important in autoimmune diseases like type 1 diabetes and have also been implicated in acute lung injury caused by infection with SARS-CoV-2. Targeting NOX enzymes directly or through scavenging free radicals may be useful therapies for autoimmunity and acute lung injury where oxidative stress contributes to pathology.

Partners in Crime: Beta-Cells and Autoimmune Responses Complicit in Type 1 Diabetes Pathogenesis.

Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of insulin-producing pancreatic beta-cells. Loss of beta-cells leads to insulin insufficiency and hyperglycemia, with patients eventually requiring lifelong insulin therapy to maintain normal glycemic control. Since T1D has been historically defined as a disease of immune system dysregulation, there has been little focus on the state and response of beta-cells and how they may also contribute to their own demise. Major hurdles to identifying a cure for T1D include a limited understanding of disease etiology and how functional and transcriptional beta-cell heterogeneity may be involved in disease progression. Recent studies indicate that the beta-cell response is not simply a passive aspect of T1D pathogenesis, but rather an interplay between the beta-cell and the immune system actively contributing to disease. Here, we comprehensively review the current literature describing beta-cell vulnerability, heterogeneity, and contributions to pathophysiology of T1D, how these responses are influenced by autoimmunity, and describe pathways that can potentially be exploited to delay T1D.

Optical sensor arrays designed for guided manufacture of perfluorocarbon nanoemulsions with a non-synthetic stabilizer.

Hydrophobic drugs are incorporated into oil-in-water nanoemulsions (OIW) either as new formulations or repurposed for intravenous delivery. Typically, these are manufactured through stepwise processes of sonication or high-pressure homogenization (HPH). The guiding criteria for most nanoemulsion manufacture are the size and homogeneity/polydispersity of the drug-laden particles with strict requirements for clinical injectables. To date, most formulation optimization is done through trial and error with stepwise sampling during processing utilizing dynamic light scattering (DLS), light obscuration sensing (LOS) or laser particle tracking (LPT) to assess manufacturing progress. The objective of this work was to develop and implement an in-line optical turbidity/nephelometry sensor array for the longitudinal in-process monitoring of nanoemulsion manufacture. A further objective was the use of this sensor array to rapidly optimize the manufacture of a sub-120 nm oxygen carrying perfluorocarbon nanoemulsion with a non-synthetic stabilizer. During processing, samples were taken for particle size measurement and further characterization. There was a significant correlation and agreement between particle size and sensor signal as well as improved process reproducibility through sensor-guided manufacture. Given the cost associated with nanoemulsion development and scale-up manufacture, our sensor arrays could be an invaluable tool for efficient and cost-effective drug development. Sensor-guided manufacturing was used to optimize oxygen-carrying nanoemulsions. These were tested, in vitro, for their ability to improve the viability of encapsulated endocrine clusters (mouse insulinoma, Min6) and to eliminate hypoxia due to oxygen mass transfer limitations. The nanomulsions significantly improved encapsulated cluster viability and reduced hypoxia within the microcapsule environment. STATEMENT OF SIGNIFICANCE: Nanoemulsions are rapidly becoming vehicles for the controlled release delivery of both hydrophilic and hydrophobic drugs given their large surface area for exchange. As work shifts from bench to large scale manufacturing, there is a critical need for technologies that can monitor and accumulate data during processing, particularly regarding the endpoint criteria of particle size and stability. To date, no such technology has been implemented in nanoemulsion manufacture. In this paper we develop and implement an optical sensor array for in-line nanoemulsion process monitoring and then use the array to optimize the development and manufacture of novel reproducible oxygen carrying nanoemulsions lacking synthetic surfactants.

Targeting Mitochondrial-Derived Reactive Oxygen Species in T Cell-Mediated Autoimmune Diseases.

Mitochondrial dysfunction resulting in oxidative stress could be associated with tissue and cell damage common in many T cell-mediated autoimmune diseases. Autoreactive CD4 T cell effector subsets (Th1,Th17) driving these diseases require increased glycolytic metabolism to upregulate key transcription factors (TF) like T-bet and RORγt that drive differentiation and proinflammatory responses. However, research in immunometabolism has demonstrated that mitochondrial-derived reactive oxygen species (ROS) act as signaling molecules contributing to T cell fate and function. Eliminating autoreactive T cells by targeting glycolysis or ROS production is a potential strategy to inhibit autoreactive T cell activation without compromising systemic immune function. Additionally, increasing self-tolerance by promoting functional immunosuppressive CD4 T regulatory (Treg) cells is another alternative therapeutic for autoimmune disease. Tregs require increased ROS and oxidative phosphorylation (OxPhos) for Foxp3 TF expression, differentiation, and anti-inflammatory IL-10 cytokine synthesis. Decreasing glycolytic activity or increasing glutathione and superoxide dismutase antioxidant activity can also be beneficial in inhibiting cytotoxic CD8 T cell effector responses. Current treatment options for T cell-mediated autoimmune diseases such as Type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) include global immunosuppression, antibodies to deplete immune cells, and anti-cytokine therapy. While effective in diminishing autoreactive T cells, they can also compromise other immune responses resulting in increased susceptibility to other diseases and complications. The impact of mitochondrial-derived ROS and immunometabolism reprogramming in autoreactive T cell differentiation could be a potential target for T cell-mediated autoimmune diseases. Exploiting these pathways may delay autoimmune responses in T1D.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

The Importance of Proper Oxygenation in 3D Culture.

Cell culture typically employs inexpensive, disposable plasticware, and standard humidified CO2/room air incubators (5% CO2, ∼20% oxygen). These methods have historically proven adequate for the maintenance of viability, function, and proliferation of many cell types, but with broad variation in culture practices. With technological advances it is becoming increasingly clear that cell culture is not a "one size fits all" procedure. Recently, there is a shift toward comprehension of the individual physiological niches of cultured cells. As scale-up production of single cell and 3D aggregates for therapeutic applications has expanded, researchers have focused on understanding the role of many environmental metabolites/forces on cell function and viability. Oxygen, due to its role in cell processes and the requirement for adequate supply to maintain critical energy generation, is one such metabolite gaining increased focus. With the advent of improved sensing technologies and computational predictive modeling, it is becoming evident that parameters such as cell seeding density, culture media height, cellular oxygen consumption rate, and aggregate dimensions should be considered for experimental reproducibility. In this review, we will examine the role of oxygen in 3D cell culture with particular emphasis on primary islets of Langerhans and stem cell-derived insulin-producing SC-ß cells, both known for their high metabolic demands. We will implement finite element modeling (FEM) to simulate historical and current culture methods in referenced manuscripts and innovations focusing on oxygen distribution. Our group and others have shown that oxygen plays a key role in proliferation, differentiation, and function of these 3D aggregates. Their culture in plastic consistently results in core regions of hypoxia/anoxia exacerbated by increased media height, aggregate dimensions, and oxygen consumption rates. Static gas permeable systems ameliorate this problem. The use of rotational culture and other dynamic culture systems also have advantages in terms of oxygen supply but come with the caveat that these endocrine aggregates are also exquisitely sensitive to mechanical perturbation. As recent work demonstrates, there is a strong rationale for the use of alternate in vitro systems to maintain physio-normal environments for cell growth and function for better phenotypic approximation of in vivo counterparts.

Auranofin-Mediated NRF2 Induction Attenuates Interleukin 1 Beta Expression in Alveolar Macrophages.

BACKGROUND: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1ß expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis. METHODS: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2. RESULTS: Expression of IL-1ß, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1ß expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1ß. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II. CONCLUSIONS: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1ß synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1ß directly inhibits transcription of the IL1ß gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation.

ICOS ligand and IL-10 synergize to promote host-microbiota mutualism.

Genome-wide association studies have identified ICOSLG, which encodes the inducible costimulator ligand (ICOSLG or ICOSL) as a susceptibility locus for inflammatory bowel disease. ICOSL has been implicated in the enhancement of pattern recognition receptor signaling in dendritic cells, induction of IL-10 production by CD4 T cells, and the generation of high-affinity antibodies to specific antigens-all of which can potentially explain its involvement in gastrointestinal inflammation. Here, we show that murine ICOSL deficiency results in significant enrichment of IL-10-producing CD4 T cells particularly in the proximal large intestine. Transient depletion of IL-10-producing cells from adult ICOSL-deficient mice induced severe colonic inflammation that was prevented when mice were first treated with metronidazole. ICOSL-deficient mice displayed reduced IgA and IgG antibodies in the colon mucus and impaired serum antibody recognition of microbial antigens, including flagellins derived from mucus-associated bacteria of the Lachnospiraceae family. Confirming the synergy between ICOSL and IL-10, ICOSL deficiency coupled with CD4-specific deletion of the Il10 gene resulted in juvenile onset colitis that was impeded when pups were fostered by ICOSL-sufficient dams. In this setting, we found that both maternally acquired and host-derived antibodies contribute to the life anti-commensal antibody repertoire that mediates this protection in early life. Collectively, our findings reveal a partnership between ICOSL-dependent anti-commensal antibodies and IL-10 in adaptive immune regulation of the microbiota in the large intestine. Furthermore, we identify ICOSL deficiency as an effective platform for exploring the functions of anti-commensal antibodies in host-microbiota mutualism.