Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs.

ObjectiveThe nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic beta-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.MethodsWe developed a model designed to "lock" lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.ResultsContinuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.ConclusionOur data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment

Microstructure and mechanical properties of 5.8 GHz microwave-sintered ZrO2/Al2O3 ceramics

[EN] Aim of the present study is to sinter zirconia nanocomposite powders doped with ceria and toughened with alumina (10Ce-TZP/Al2O3) by non-conventional means, i.e. microwave sintering technology. The sintering effects of various microwave applicators and frequency generators were evaluated using an optimised experimental set-up. The microwave-sintered samples were compared with the composites sintered by the conventional method. The mechanical properties of the ceramic composites were evaluated by their hardness, fracture toughness and Young's modulus. Likewise, their density and microstructure were analysed.The authors thank the Generalitat Valenciana for the financial support provided to the PROMETEU/2016/040 project. A. Borrell is grateful to the Spanish Ministry of Economy and Competitiveness for her RyC contract (RYC-2016-20915).Gil-Flores, L.; Salvador Moya, MD.; Penaranda-Foix, FL.; Fernández, A.; Suárez-Menéndez, M.; Rosa, R.; Veronesi, P.... (2019). Microstructure and mechanical properties of 5.8 GHz microwave-sintered ZrO2/Al2O3 ceramics. Ceramics International. 45(14):18059-18064. https://doi.org/10.1016/j.ceramint.2019.06.026S1805918064451

Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCerBf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCerBf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis

Therapeutic Interventions to Modulate the Anti-Islet Response in Autoimmune Diabetes

Autoimmunity is caused by a loss of tolerance and results in an imbalance in immune homeostasis, particularly regulatory versus effector activities against self-antigens. Therapeutic approaches for the treatment of autoimmune diseases need to attack key components of the immune response to restore this balance. In this body of work I have worked toward this end using three distinct approaches to in the context of type 1 diabetes using the non-obese diabetic mouse model. First, I used a global approach to shift the balance of regulatory to effector T cells. I show that non-Fc receptor binding anti-CD3 monoclonal antibody treatment, which has been previously shown to reserve diabetes, alters the ratio of effector to regulatory T cells (Tregs) due to preferential depletion of activated effector T cells. In addition, treatment with anti-CD3 mAbs leads to increased expression of Helios in Tregs, suggesting stabilization of Tregs may account to the extended efficacy of this antibody. Second, I used FTY720 to "lock" lymphocytes in the lymph node and pancreas thereby isolating these locations. I show that continuous treatment with FTY720 prevents diabetes development but treatment withdrawal leads to rapid onset of disease. Furthermore, morphological changes that occur during the development of the disease in control and treated mice show that tertiary lymphoid organ development and their subsequent destruction correlate with disease progression suggesting that islet destruction maybe be due to loss of TLO integrity. Finally, I used a targeted approach to directly alter memory T cells. I show that treatment of NOD mice with anti-IL-7Rα monoclonal antibodies prevents and cures diabetes and induces expression of the inhibitory receptor PD-1 on memory T cells. Moreover, inhibiting the interaction of PD-1 with its ligand PD-L1 restores disease in cured mice. The data suggest that IL-7 contributes to the pathogenesis of autoimmune diabetes by keeping memory T cells in a functionally competent, tolerance-resistant state, and uncover a novel link between IL-7 and the PD-1/PD-L1 tolerance pathway. Together the data reveal biological the mechanisms at the basis of therapeutic interventions aimed at preventing or reversing diabetes through manipulating the immunological pathways underling the pathogenesis of the disease. The insights will improve our ability to translate these interventions to the treatment of patients with type 1 diabetes

Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs.

ObjectiveThe nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic beta-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.MethodsWe developed a model designed to "lock" lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.ResultsContinuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.ConclusionOur data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment

Aberrant innate immune activation following tissue injury impairs pancreatic regeneration.

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury