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Summary: An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of β-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human β-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.

IntroductionType 1 diabetes (T1D) is defined by immune cell infiltration of the pancreas, in particular the islets of Langerhans, referred to as insulitis, which is especially prominent during the early disease stages in association with decreased beta cell mass. An in-depth understanding of the dynamics and phenotype of the immune cells infiltrating the pancreas and the accompanying changes in their profiles in peripheral blood during T1D development is critical to generate novel preventive and therapeutic approaches, as well as to find biomarkers for the disease process.MethodsUsing multi-parameter flow cytometry, we explored the dynamic changes of immune cells infiltrating the pancreas and the pancreatic draining lymph nodes (PLN), compared to those in peripheral blood in female and male non-obese diabetic (NOD) mice during T1D progression.ResultsThe early stages of T1D development were characterized by an influx of innate dendritic cells and neutrophils in the pancreas. While dendritic cells seemed to move in and out (to the PLN), neutrophils accumulated during the pre-symptomatic phase and reached a maximum at 8 weeks of age, after which their numbers declined. During disease progression, CD4+ and CD8+ T cells appeared to continuously migrate from the PLN to the pancreas, which coincided with an increase in beta cell autoimmunity and insulitis severity, and a decline in insulin content. At 12 weeks of age, CD4+ and especially CD8+ T cells in the pancreas showed a dramatic shift from naïve to effector memory phenotype, in contrast to the PLN, where most of these cells remained naïve. A large proportion of pancreas infiltrating CD4+ T cells were naïve, indicating that antigenic stimulation was not necessary to traffic and invade the pancreas. Interestingly, a pre-effector-like T cell dominated the peripheral blood. These cells were intermediates between naïve and effector memory cells as identified by single cell RNA sequencing and might be a potential novel therapeutic target.ConclusionThese time- and tissue-dependent changes in the dynamics and functional states of CD4+ and CD8+ T cells are essential steps in our understanding of the disease process in NOD mice and need to be considered for the interpretation and design of disease-modifying therapies.

Abstract Background In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. Methods Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. Results VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig’s within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig’s. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig’s, anti-dsDNA Ig’s and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. Conclusion During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. Trail registration ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

BackgroundRestoration of immune tolerance to disease-relevant antigens is an appealing approach to prevent or arrest an organ-specific autoimmune disease like type 1 diabetes (T1D). Numerous studies have identified insulin as a key antigen of interest to use in such strategies, but to date, the success of these interventions in humans has been inconsistent. The efficacy of antigen-specific immunotherapy may be enhanced by optimising the dose, timing, and route of administration, and perhaps by the inclusion of adjuvants like alum. The aim of our study was to evaluate the effect of an insulin peptide vaccine formulated with alum to prevent T1D development in female non-obese diabetic (NOD) mice when administered during late-stage pre-diabetes.MethodsStarting at 10 weeks of age, female NOD mice received four weekly subcutaneous injections of an insulin B:8-24 (InsB:8-24) peptide with (Ins+alum) or without Imject® alum (Ins) as adjuvant. Diabetes incidence was assessed for up to 30 weeks of age. Insulin autoantibodies and C-peptide concentrations were measured in plasma and flow cytometric analysis was performed on pancreatic-draining lymph nodes (PLN) and pancreas using an InsB:12-20-reactive tetramer.ResultsInsB:8-24 peptide formulated in alum reduced diabetes incidence (39%), compared to mice receiving the InsB:8-24 peptide without alum (71%, P < 0.05), mice receiving alum alone (76%, P < 0.01), or mice left untreated (70%, P < 0.01). This was accompanied by reduced insulitis severity, and preservation of C-peptide. Ins+alum was associated with reduced frequencies of pathogenic effector memory CD4+ and CD8+ T cells in the pancreas and increased frequencies of insulin-reactive FoxP3+ Tregs in the PLN. Of interest, insulin-reactive Tregs were enriched amongst populations of Tregs expressing markers indicative of stable FoxP3 expression and enhanced suppressive function.ConclusionAn InsB:8-24 peptide vaccine prevented the onset of T1D in late-stage pre-diabetic NOD mice, but only when formulated in alum. These findings support the use of alum as adjuvant to optimise the efficacy of antigen-specific immunotherapy in future trials.

Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol–myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease.

Background and aims: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a T-cell-mediated destruction of the pancreatic insulin-producing beta cells. A growing body of evidence suggests that abnormalities in neutrophils and neutrophil extracellular trap (NET) formation (NETosis) are associated with T1D pathophysiology. However, little information is available on whether these changes are primary neutrophil defects or related to the environmental signals encountered during active disease. Methods: In the present work, the NET proteome (NETome) of phorbol 12-myristate 13-acetate (PMA)- and ionomycin-stimulated neutrophils from people with established T1D compared to healthy controls (HC) was studied by proteomic analysis. Results: Levels of NETosis, in addition to plasma levels of pro-inflammatory cytokines and NET markers, were comparable between T1D and HC subjects. However, the T1D NETome was distinct from that of HC in response to both stimuli. Quantitative analysis revealed that the T1D NETome was enriched in proteins belonging to metabolic pathways (i.e., phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase). Complementary metabolic profiling revealed that the rate of extracellular acidification, an approximate measure for glycolysis, and mitochondrial respiration were similar between T1D and HC neutrophils in response to both stimuli. Conclusion: The NETome of people with established T1D was enriched in metabolic proteins without an apparent alteration in the bio-energetic profile or dysregulated NETosis. This may reflect an adaptation mechanism employed by activated T1D neutrophils to avoid impaired glycolysis and consequently excessive or suboptimal NETosis, pivotal in innate immune defence and the resolution of inflammation.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5’-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

Summary: The identification and validation of circulating small non-coding RNA (sncRNA) as biomarkers for disease diagnosis, staging, and response to novel therapies is still a compelling challenge. Pre-analytical variables, such as storage temperature or blood hemolysis, and different analytical approaches affect sncRNA stability, detection, and expression, resulting in discrepancies among studies. Here, we report a systematic standardized protocol to reproducibly analyze circulating sncRNAs, employing high-throughput sncRNA sequencing and qRT-PCR validation, from 200 μL of human plasma samples.For details on the use and execution of this protocol, please refer to Ventriglia et al. (2020), Sebastiani et al. (2017), and Dotta et al. (2018).

Summary: The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (19F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of 19F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression. : Infection Control in Health Technology; Medical Imaging; Mycology Subject Areas: Infection Control in Health Technology, Medical Imaging, Mycology

This article contains data related to the inflammatory cytokine and investigated pathways involved in bacterial clearance reported in “Airway infection with Nontypeable Haemophilus influenzae is more rapidly eradicated in vitamin D deficient mice” (Serré et al., 2018) [1]. Vitamin D deficient or sufficient mice were oropharyngeally instilled with 106 NTHi and sacrificed at 4, 8, 24 and 72 h post-infection. We measured proinflammatory cytokines (KC, TNF-α, IL-1β, IL6 and MCP-1) markers of bacterial clearance pathways (myeloid peroxidase, nitric oxide, complement C5a and immunoglobulin A) in bronchoalveolar fluid (BALF) during infection and mRNA expression levels of innate immune defense mechanism markers (mucin glycoproteins, pathogen recognitions receptor TLR2 and TLR4, antimicrobial peptides SLPI, REG3γ, lysozyme, BD-1, BD-2, BD-3 and surfactant proteins SP-A and SP-D) in lung homogenate. Finally, genomic DNA of NTHi (protein D) measured in lung homogenate was used as an indicator of NTHi invasion of alveolar macrophages or epithelial cells.

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human β-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the β-cell line EndoC-βH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β-cell virus tropism.

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12–20 or InsB13–2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.

Steviol glycosides are sweet-tasting compounds isolated from a South American shrub and are increasingly used as sweeteners in foods and beverages. Philippaertet al. demonstrate that steviol glycosides potentiate Ca2+-dependent TRPM5 activity and promote glucose-induced insulin secretion and glucose tolerance.

The presence of islet cells double positive for insulin and glucagon (Ins+/Glu+) has been described in the pancreas from both type 2 (T2D) and type 1 (T1D) diabetic subjects. We studied the role of pro-inflammatory cytokines on the occurrence, trajectory, and characteristics of Ins+/Glu+ cells in human pancreatic islets. Pancreas samples, isolated islets, and dispersed islet cells from 3 T1D and 11 non-diabetic (ND) multi-organ donors were studied by immunofluorescence, confocal microscopy, and/or electron microscopy. ND islet cells were exposed to interleukin-1β and interferon-γ for up to 120 h. In T1D islets, we confirmed an increased prevalence of Ins+/Glu+ cells. Cytokine-exposed islets showed a progressive increase of Ins+/Glu+ cells that represented around 50% of endocrine cells after 120h. Concomitantly, cells expressing insulin granules only decreased significantly over time, whereas those containing only glucagon granules remained stable. Interestingly, Ins+/Glu+ cells were less prone to cytokine-induced apoptosis than cells containing only insulin. Cytokine-exposed islets showed down-regulation of β-cell identity genes. In conclusion, pro-inflammatory cytokines induce Ins+/Glu+ cells in human islets, possibly due to a switch from a β- to a β-/α-cell phenotype. These Ins+/Glu+ cells appear to be resistant to cytokine-induced apoptosis.

Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes.

Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH)2D3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH)2D3, hypoxia did not change the tolerogenic function of 1,25(OH)2D3-treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH)2D3-conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH)2D3, because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.

Previous work has revealed that Cx36, the sole connexin expressed in the insulin-producing beta cells, enhances the secretion of insulin, and promotes the resistance of beta cells against pro-inflammatory cytokines. In parallel, the anti-diabetic sulphonylurea glibenclamide was shown to promote the assembly and function of Cx36 channels. Here, we assessed whether glibenclamide could protect the insulin-producing cells against conditions mimicking those expected at the onset of type 1 diabetes. We found that the drug 1) protected in vitro the mouse MIN6 cells from the apoptosis and loss of Cx36, which are induced by Th1 cytokines; 2) prevented the development of hyperglycemia as well as the loss of beta cells and Cx36, which rapidly develop with aging in untreated NOD mice; 3) modified the proportion of effector CD4+ and CD8+ T cells in pancreatic draining lymph nodes. The data imply that an early glibenclamide treatment may help protecting beta cells against the autoimmune attack, which triggers the development of type 1 diabetes.

Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)2D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)2D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)2D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)2D did not improve defects in phagocytosis of E. coli bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)2D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.

The presence of pancreatic duct cells in clinical islet grafts may affect long-term metabolic success. Human pancreatic duct cells express factors that may exert both protective and damaging effects on islet cells in the graft. Here we studied the potential of commonly used immunosuppressive drugs in islet transplantation—sirolimus, tacrolimus, and mycophenolate mofetil (MMF)—to influence the inflammatory and angiogenic capacity of human pancreatic duct cells in addition to their proliferation and reprogramming abilities. Our data show that the expression of specific proinflammatory cytokines by the human pancreatic duct cells was either unaltered or inhibited by the immunosuppressants studied, especially tacrolimus and MMF, whereas expression of chemotactic and angiogenic factors was unaffected. Although none of the immunosuppressants directly led to duct cell death, MMF prevented duct cell proliferation, and sirolimus inhibited neurogenin 3-mediated duct-to-(neuro)endocrine cell reprogramming. Our data indicate that the immunosuppressant tacrolimus was the least aggressive on the angiogenic, proliferative, and reprogramming potential of human pancreatic duct cells, while it was most powerful in inhibiting inflammatory cytokines, which may influence the outcome of islet transplantation.

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+ CD25high CD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+ CD25high CD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+ CD25high CD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.

β-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting β-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and β-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional β-cell like cells may serve to gain insight into signals that govern β-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful β-cells for cell therapy of T1D.

Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased β- and δ- but not α-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.