Ubiquitin D regulates IRE1 α/c-Jun N-terminal kinase (JNK) protein-dependent apoptosis in pancreatic beta cells

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1 alpha is critical for this transition. IRE1 alpha activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1 alpha-interacting protein ubiquitin D (UBD) on the regulation of IRE1 alpha and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1 alpha interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown ofUBDin human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1 alpha activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1 beta and interferon (IFN)-gamma in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1 alpha in human and rodent beta cells, modulating IRE1 alpha-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1 alpha/JNK pro-apoptotic pathway in cytokine-exposed beta cells

Autoimmunity against INS-IGF2 expressed in human pancreatic islets.

Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine 1) expression of INS-IGF2 in human pancreatic islets and 2) autoantibodies in newly diagnosed type 1 diabetes children and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared to donors with either type 2 diabetes (p=0.006) or high HbA1c levels (p<0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed type 1 diabetes patients (n=304) compared to healthy controls (n=355; p<0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes

A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells

Novel insights in antimicrobial and immunomodulatory mechanisms of action of PepBiotics CR-163 and CR-172

Objectives: Our group recently developed a new group of antimicrobial peptides termed PepBiotics, of which peptides CR-163 and CR-172 showed optimized antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus without inducing antimicrobial resistance. In this study, the antibacterial mechanism of action and the immunomodulatory activity of these two PepBiotics was explored. Methods: RAW264.7 cells were used to determine the ability of PepBiotics to neutralize Lipopolysaccharide (LPS)-and Lipoteichoic acid (LTA)-induced activation of macrophages. Isothermal titration calorimetry and competition assays with dansyl-labeled polymyxin B determined binding characteristics to LPS and LTA. Combined bacterial killing with subsequent macrophage activation assays was performed to determine so-called ‘silent killing’. Finally, flow cytometry of peptide-treated genetically engineered Escherichia coli expressing Green Fluorescent Protein (GFP) and mCherry in the cytoplasm and periplasm, respectively, further established the antimicrobial mechanism of PepBiotics. Results: Both CR-163 and CR-172 were shown to have broad-spectrum activity against ESKAPE pathogens and E. coli using a membranolytic mechanism of action. PepBiotics could exothermically bind LPS/LTA and were able to replace polymyxin B. Finally, it was demonstrated that bacteria killed by PepBiotics were less prone to stimulate immune cells, contrary to gentamicin and heat-killed bacteria that still elicited a strong immune response. Conclusions: These studies highlight the multifunctional nature of the two peptide antibiotics as both broad-spectrum antimicrobial and immunomodulator. Their ability to kill bacteria and reduce unwanted subsequent immune activation is a major advantage and highlights their potential for future therapeutic use

Rapid Insulinotropic Action of Low Doses of Bisphenol-A on Mouse and Human Islets of Langerhans: Role of Estrogen Receptor β

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of polycarbonate plastics. It alters pancreatic β-cell function and can be considered a risk factor for type 2 diabetes in rodents. Here we used ERβ−/− mice to study whether ERβ is involved in the rapid regulation of KATP channel activity, calcium signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of BPA in β-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased KATP channel activity, increased glucose-induced [Ca2+]i signals and insulin release in β-cells from WT mice but not in cells from ERβ−/− mice. The rapid reduction in the KATP channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that BPA behaves as a strong estrogen via nuclear ERβ and indicate that results obtained with BPA in mouse β-cells may be extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans

Estrogen and Serotonin – old dogs, new tricks, Implications for pancreatic beta-cell function

Islet hormone secretion is tightly regulated by metabolic status as well as local and circulating factors. These factors can activate different receptors on the pancreatic islet cells, for instance G-protein coupled receptors (GPCRs). When activated, these receptors are able to fine-tune islet hormone secretion and regulate overall β-cell function. Estrogen and serotonin are circulating factors that bind to GPCRs. First, we studied the activation of GPER-1 in pancreatic islets using two agonists G-1 and Estrogen (E2). Both G-1 and E2 displayed a similar response in mouse and human islets even in the presence of estrogen receptor blockers, ICI 182, 720 and EM652. G-1 and E2 potentiated insulin secretion and inhibited glucagon and somatostatin secretion. G-1 induced cAMP generation, suggesting positive coupling to adenylate cyclase and a subsequent rise in insulin release. Furthermore both agonists protected pancreatic islets from cytokine-induced apoptosis via activation of anti-apoptotic signals, CREB, ERK1/2 and AKT and reduced phosphorylation of the pro-apoptotic signals SAPK/JNK and p38. Second, we studied serotonin (5-HT) receptors in human islets and INS (832/13) cells. We detected 15 different 5-HT receptors and the 5-HT producing enzymes, TPH1 and TPH2 as well as DDC. Cellular localization for 5-HT1A, 5-HT1D and 5-HT2A were observed in both β- and α-cells; while 5-HT2B was only present in β-cells. Agonists targeting these four receptors were able to either inhibit or stimulate insulin secretion from human islets and INS (832/13) cells. In addition, 5-HT was quantified using GC/MS in INS (832/13) cells, rat islets and detected in human α and β-cells with immunohistochemistry. Third, we investigated the peripheral role of a 5-HT2 receptor agonist, α-methyl serotonin maleate salt (AMS) in insulin resistance and β cell function. Long-term treatment with AMS in a high fat diet fed mouse model resulted in increased insulin sensitivity in vivo in high fat fed AMS treated mice. Moreover, insulin secretion from AMS treated control fed mice in vitro was decreased while plasma glucose levels were similar in vivo between AMS treated and untreated controls. In addition, AMS mediated protection from lipotoxicity in INS-1(832/13) cells. In conclusion, this thesis contributes to increased understanding of how estrogen and peripheral 5-HT mediate their effects on islet function and overall glucose homeostasis

Interplay between Outer Membrane Vesicles and Host Defense Peptides : Bacterial shields versus Host swords

The scope of this thesis is the interplay between Host Defense Peptides (HDPs) and Outer Membrane Vesicles (OMVs). HDPs have both antimicrobial and immunomodulatory properties. OMVs contain multiple antigens in their native environment and are therefore promising for vaccine development. It was also thought that OMVs could protect bacteria against killing by HDPs, but additionally it was investigated whether OMV release could be a response of bacteria upon HDPs in the environment. This increase in OMV yield could also be used for vaccine production, if OMV properties are not significantly altered. HDPs are known to have membrane-active properties, but the role of LPS-binding in killing of Gram-negative bacteria is not yet evident. Therefore, LPS-binding was correlated with HDP antibacterial effectivity, to distinguish between LPS acting as anchor and facilitating HDP functions or LPS acting as a sink and inhibiting HDP functions. Furthermore, immunomodulatory capabilities of HDPs were investigated, since this neutralization has not yet been described for OMV-induced activation. If HDPs could balance OMV-induced immune responses, the combination could be promising for vaccine development. In chapter 3, it was shown that OMVs can indeed act as a bacterial defense against HDPs. Not only were OMV quantities increased after stimulation with sub-lethal concentrations of HDPs, but also could addition of isolated OMVs to bacterial cultures protect against HDP killing. However, this was not true for all HDPs tested. The differences in the mechanisms of action of HDPs were further elucidated in chapter 4. CATH-2 and PMAP-36 were shown to be membrane active, but their effectivity did not correlate with LPS-binding. PMAP-23 showed an interaction with LPS and probably acts via a carpet model, but antibacterial effectivity was not correlated with LPS affinity. PR-39 was shown to be purely intracellularly active and to not affect bacterial membranes at all but did show binding to LPS. Stronger LPS-binding even correlated with enhanced bacterial killing for PR-39. Not only HDPs were shown to affect bacterial membranes, but also heat was shown to affect bacteria and enhance OMV release. In chapter 5, PMAP-36 was studied in more detail with respect to OMV induction and also to the immunomodulation of OMVs. Induction with PMAP-36 resulted in the presence of the peptide in isolated OMVs but this presence did not affect immunomodulation. When PMAP-36 was added to spontaneously formed OMVs (sOMVs) after isolation, it did show a neutralizing effect. Therefore, a large array of HDPs was investigated for their immunomodulatory capabilities in combination with OMVs (chapter 6). Four out of the eight HDPs tested showed immunomodulatory effects, being LL-37, CATH-2, PMAP-36 and K9CATH. They were further investigated for their specific TLR neutralizing capabilities. This revealed that OMVs were not only able to stimulate TLR4 and TLR2, but also TLR5 and TLR9. TLR neutralization was HDP specific, but TLR5 was consistently not neutralized by any peptide tested. Concluding, OMVs indeed play a role in defense against HDPs and HDPs are able to modulate OMV-induced immune responses. However, LPS-binding did not seem to correlate with HDP antibacterial activity

Interplay between Outer Membrane Vesicles and Host Defense Peptides : Bacterial shields versus Host swords

The scope of this thesis is the interplay between Host Defense Peptides (HDPs) and Outer Membrane Vesicles (OMVs). HDPs have both antimicrobial and immunomodulatory properties. OMVs contain multiple antigens in their native environment and are therefore promising for vaccine development. It was also thought that OMVs could protect bacteria against killing by HDPs, but additionally it was investigated whether OMV release could be a response of bacteria upon HDPs in the environment. This increase in OMV yield could also be used for vaccine production, if OMV properties are not significantly altered. HDPs are known to have membrane-active properties, but the role of LPS-binding in killing of Gram-negative bacteria is not yet evident. Therefore, LPS-binding was correlated with HDP antibacterial effectivity, to distinguish between LPS acting as anchor and facilitating HDP functions or LPS acting as a sink and inhibiting HDP functions. Furthermore, immunomodulatory capabilities of HDPs were investigated, since this neutralization has not yet been described for OMV-induced activation. If HDPs could balance OMV-induced immune responses, the combination could be promising for vaccine development. In chapter 3, it was shown that OMVs can indeed act as a bacterial defense against HDPs. Not only were OMV quantities increased after stimulation with sub-lethal concentrations of HDPs, but also could addition of isolated OMVs to bacterial cultures protect against HDP killing. However, this was not true for all HDPs tested. The differences in the mechanisms of action of HDPs were further elucidated in chapter 4. CATH-2 and PMAP-36 were shown to be membrane active, but their effectivity did not correlate with LPS-binding. PMAP-23 showed an interaction with LPS and probably acts via a carpet model, but antibacterial effectivity was not correlated with LPS affinity. PR-39 was shown to be purely intracellularly active and to not affect bacterial membranes at all but did show binding to LPS. Stronger LPS-binding even correlated with enhanced bacterial killing for PR-39. Not only HDPs were shown to affect bacterial membranes, but also heat was shown to affect bacteria and enhance OMV release. In chapter 5, PMAP-36 was studied in more detail with respect to OMV induction and also to the immunomodulation of OMVs. Induction with PMAP-36 resulted in the presence of the peptide in isolated OMVs but this presence did not affect immunomodulation. When PMAP-36 was added to spontaneously formed OMVs (sOMVs) after isolation, it did show a neutralizing effect. Therefore, a large array of HDPs was investigated for their immunomodulatory capabilities in combination with OMVs (chapter 6). Four out of the eight HDPs tested showed immunomodulatory effects, being LL-37, CATH-2, PMAP-36 and K9CATH. They were further investigated for their specific TLR neutralizing capabilities. This revealed that OMVs were not only able to stimulate TLR4 and TLR2, but also TLR5 and TLR9. TLR neutralization was HDP specific, but TLR5 was consistently not neutralized by any peptide tested. Concluding, OMVs indeed play a role in defense against HDPs and HDPs are able to modulate OMV-induced immune responses. However, LPS-binding did not seem to correlate with HDP antibacterial activity