Klaus Pechhold

No Evidence for Mouse Pancreatic β-Cell Epithelial-Mesenchymal Transition In Vitro

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic β-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet “progenitor” cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse β-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from β-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal “progenitors” might not represent the best possible source for generation of physiologically competent β-cells for treatment of diabetes.

Low Dose Streptozotocin-Induced Diabetes in Rat Insulin Promoter-mCD80-Transgenic Mice Is T Cell Autoantigen-Specific and CD28 Dependent

Although transgenic mice expressing murine B7-1 (mCD80) on their pancreatic β cells under the rat insulin-1 promoter (RIP-mCD80+ mice) rarely develop spontaneous β cell destruction and diabetes, we have previously reported the transgene-dependent induction of profound insulitis and lethal diabetes following multiple low dose injections of the β cell toxin streptozotocin (MLDS) in RIP-mCD80+ mice. Here, we have further characterized this MLDS-induced diabetes model using the RIP-mCD80+ mice and now demonstrate that disease is critically dependent on T cell signaling via CD28. Thus, although naive RIP-mCD80+ and nontransgenic littermates have comparable gross β cell mass, and immediately following MLDS induction the mice display similar degrees of insulitis and decrements in the β cell mass, only transgenic mice continued to destroy their β cells and develop insulin-dependent diabetes mellitus. Strikingly, MLDS-induced diabetes was completely prevented in CD28-deficient mice (RIP-mCD80+CD28−/−) due to abrogation of leukocytes infiltrating their pancreatic islets. We further characterized MLDS-induced diabetes in the RIP-mCD80+ mice by demonstrating that the MLDS-induced lymphocytic islet infiltrate contained a substantial frequency of autoantigen-specific, IFN-γ-secreting, CD8+ T cells. We conclude that MLDS-induced β cell destruction and subsequent insulin-dependent diabetes mellitus in RIP-mCD80+ mice is T cell-mediated as it involves both Ag-specific recognition of self-target molecules in the inflamed pancreatic islet (signal 1) and is CD28 costimulation dependent (signal 2).

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection

Exploring the pathophysiology underlying diabetes mellitus requires characterizing the cellular constituents of pancreatic islets, primarily insulin-producing β-cells. Such efforts have been limited by inadequate techniques for purifying islet cellular subsets for further biochemical and gene-expression studies. Using intracytoplasmic staining and fluorescence-activated cell-sorting (FACS) followed by quantitative nuclease protection assay (qNPA™) technology, we examined 30 relevant genes expressed by islet subpopulations. Purified islet cell subsets expressed all four tested “housekeeping” genes with a surprising variability, dependent on both cell lineage and developmental stage, suggesting caution when interpreting housekeeping gene-normalized mRNA quantifications. Our new approach confirmed expected islet cell lineage-specific gene expression patterns at the transcriptional level, but also detected new phenotypes, including mRNA-profiles (supported by immunohistology) demonstrating that during pregnancy, some β-cells express Mafb, previously found only in immature β-cells during embryonic development. Overall, qNPA™ gene expression analysis using intracellular-stained then FACS-sorted cells has broad applications beyond islet cell biology.

Deciphering von Hippel-Lindau (VHL/Vhl)-Associated Pancreatic Manifestations by Inactivating Vhl in Specific Pancreatic Cell Populations

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in α-cells or an insulin promoter in β-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated α-cells or β-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Dynamic Changes in Pancreatic Endocrine Cell Abundance, Distribution, and Function in Antigen-Induced and Spontaneous Autoimmune Diabetes

OBJECTIVE Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by β-cell–specific killing by autoreactive T-cells. Less is known about β-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents. RESEARCH DESIGN AND METHODS We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell–mediated β-cell–destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse. RESULTS The proportion of pancreatic insulin-positive β-cells to glucagon-positive α-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe β-cell depletion accompanied by phenotypic β-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of α-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non–insulin-induced hypoglycemia. CONCLUSIONS Systematically applying multicolor flow cytometry and immunohistochemistry to track declining β-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet α-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub–islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.

Multiple Endocrine Neoplasia Type 1 Deletion in Pancreatic α-Cells Leads to Development of Insulinomas in Mice

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions. The lack of suitable cell lines to study alpha- and beta-cells interactions have led us to develop an alpha-cell-specific Cre-expressing transgenic line utilizing a glucagon promoter sequence, the Glu-Cre transgenic mouse. Here, we demonstrate that the Glu-Cre could specifically and efficiently excise floxed target genes in adult islet alpha-cells. We further showed that deletion of the tumor suppressor gene, multiple endocrine neoplasia type 1 (Men1), in alpha-cells led to tumorigenesis. However, to our surprise, the lack of Men1 in alpha-cells did not result in glucagonomas but rather beta-cell insulinomas. Because deletion of the Men1 alleles was only present in alpha-cells, our data suggested that cross communication between alpha- and beta-cells contributes to tumorigenesis in the absence of Men1. Together, we believed that the new model systems described here will allow future studies to decipher cellular interactions between islet alpha- and beta-cells in a physiological context.

Very-Low-Dose Streptozotocin Induces Diabetes in Insulin Promoter-mB7-1 Transgenic Mice

Transgenic mice that express mouse B7-1 (mB7-1, recently designated CD80) on their pancreatic β-cells maintain normal islet architecture, have normal pancreatic insulin content, and only rarely spontaneously develop insulitis and diabetes. Nevertheless, these mice display an extreme sensitivity to streptozotocin (STZ)-induced diabetes. Female mice were administered two STZ doses intraperitoneally, 20 and 40 mg/kg body wt, each for five consecutive days. Nontransgenic but otherwise syngeneic mice responded to the STZ with a moderate diminution in pancreatic insulin content but not with persistent glycosuria. In striking contrast, STZ administered to transgenic mice resulted in a severe diminution of pancreatic insulin content and in diabetes. Notably, the lower STZ dose resulted in diabetes only after a prolonged (26- to 100-day) latency. STZ-induced diabetes appears to be T-cell dependent, since treatment with T-cell–depleting (and in particular CD8+ subset-depleting) antibodies ameliorated the response. Anti-mB7-1 monoclonal antibody administration also prevented STZ-induced diabetes. Thus, unmasked mB7-1 is a required component in the pathway resulting in β-cell killing. Immunohistological analysis revealed that early after STZ administration, both mB7-1 transgenic and nontransgenic mice developed insulitis. While this insulitis resolved in the nontransgenic mice, the islet-infiltrating CD4+ and CD8+ T-cells in the transgenic mice were associated with complete β-cell destruction. These data suggest that STZ-induced diabetes in mB7-1 transgenic mice is an immune-mediated process with distinct potential advantages over existing insulindependent diabetes models.

Beta cell-specific CD80 (B7-1) expression disrupts tissue protection from autoantigen-specific CTL-mediated diabetes.

T cell responses toward pancreatic beta cell autoantigens arise spontaneously or on immunization in many mouse strains, yet sustained islet infiltration and progressive diabetes rarely ensues. Most mouse diabetes models overcome the innocuous coexistence of anti-islet specific T cells and endogenous islets via incompletely understood mechanisms (e.g. the spontaneous disease onset of the non-obese diabetic mouse) or depend on overwhelming numbers of peripheral islet-specific T cells. We report that insulin promoter murine CD80 (RIP-CD80) transgenic mice are extraordinarily susceptible to autoantigen-induced diabetes, while spontaneous disease is rare. Autoimmunity to the pancreatic beta cell-expressed glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) was elicited by a single injection of syngeneic fibroblastoid cell lines (FCL) loaded with the immunodominant LCMV-GP peptide, gp33. While both RIP-GP(+)and RIP-CD80(+)GP(+)mice mounted moderate CD4-independent CTL responses, only CD80(+)GP(+)mice developed severe insulitis and diabetes due to islet-infiltration of activated, gp33-specific, CD8(+)T cells. Strikingly, DNA immunization using plasmids encoding LCMV-GP or murine preproinsulin also efficiently induced Ag-specific RIP-CD80-dependent diabetes. We conclude that aberrant CD80-expression in a peripheral tissue disrupts that tissues natural resistance to CD8 T cell-mediated autoimmune destruction. This rodent model thus represents a novel approach to identify beta cell-derived autoantigenic determinants involved in the pathogenesis of autoimmune diabetes, and may also serve as a prototype approach to uncover relevant autoantigens leading to a variety of organ-specific autoimmune disorders.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

BackgroundPancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice.ResultsWe compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice.ConclusionAAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Cytotoxic T cell-mediated diabetes in RIP-CD80 transgenic mice: autoantigen peptide sensitivity and fine specificity.

Abstract:  Rodent immune‐mediated diabetes model studies have advanced understanding of β cell–specific T cell responses, and the testing of therapeutic approaches. We have used an inducible diabetes model based on rat insulin promotor (RIP)–driven expression of CD80 (B7‐1) on pancreatic β cells. Using these mice, we have established that immunizing with a single autoantigen can promote progressive islet inflammation and eventually T cell–mediated diabetes. We now describe a potent immunization protocol using peptide‐pulsed mature dendritic cells (DCs) to examine peptide epitopes derived from endogenous (preproinsulin) and transgenically expressed β cell antigens, namely lymphocytic choriomeningitis virus glycoprotein (LCMV‐GP). LCMV‐GP epitopes efficiently promote β cell destruction, and the autoantigenic peptide concentration used to load the DCs correlates directly with diabetes onset. The system allowed us to assess cytotoxic T cell (CTL) fine specificity by immunizing with DCs presenting altered peptide ligands (APLs) of the dominant LCMV‐GP epitope, gp33. Finally, using an adoptive transfer system, we tested alternative in vitro T cell activation conditions, including APLs and mitogens, for their impact on T cell effector function and diabetes onset. Our studies revealed a marked discrepancy between (inflammatory) effector functions and diabetes progression, thus emphasizing the importance of structural identity between sensitizing and target epitope and the context of initial T cell activation.