Apolipoprotein L genes are novel mediators of inflammation in beta cells.

AIMS/HYPOTHESIS: Inflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage. METHODS: We used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-ßH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells. RESULTS: APOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus. CONCLUSIONS/INTERPRETATION: APOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes. DATA AVAILABILITY: scRNAseq data generated by our laboratory and used in this study are available in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/ ), accession number GSE218316.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

Oxidative Stress Leads to ß-Cell Dysfunction Through Loss of ß-Cell Identity.

Pancreatic ß-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. ß-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human ß-cells. We show that primary human ß-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of ß-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced ß-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional ß-cell mass in early stages of development of T1D.

Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells.

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies.

Topologically heterogeneous beta cell adaptation in response to high-fat diet in mice.

AIMS: Beta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice. METHODS: C57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice. RESULTS: SR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation. CONCLUSIONS: HFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.

The bone marrow functions as the central site of proliferation for long-lived NK cells.

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

Immunoproteasome-deficiency has no effects on NK cell education, but confers lymphocytes into targets for NK cells in infected wild-type mice.

Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo.

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits ß5i/LMP7 and ß2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.

Effective CD8(+) T cell priming and tumor protection by enterotoxin B subunit-conjugated peptides targeted to dendritic cells.

In our previous studies we have shown that bacterial enterotoxin B subunits are effective vehicles to deliver antigen into the MHC class I processing route. Here we have used the non-toxic Escherichia coli heat labile enterotoxin B subunit (EtxB) conjugated to OVA peptide (EtxB-peptide) to address the impact on induction of specific CD8(+) T cells in vivo. Although incubation of DCs with these EtxB-peptide conjugates as such did not induce DC maturation in vitro MHC class I antigen presentation was much more efficient as compared to peptide alone. Antigen presentation was further enhanced upon DC maturation with the TLR-4 ligand LPS. Injection of matured DCs incubated with EtxB-peptide conjugates lead to strong induction of OVA-specific CD8(+) T lymphocytes and fully prevented the outgrowth of lethal B16 melanoma in wild type mice. Our data demonstrate that bacterial non-toxic B subunit-peptide conjugates are potent vaccine vehicles for induction of protective CD8(+) T cell responses.

The proteasome immunosubunit multicatalytic endopeptidase complex-like 1 is a T-cell-intrinsic factor influencing homeostatic expansion.

Homeostatic regulatory mechanisms maintain the constant ratios between different lymphocyte subsets in the secondary lymphoid organs. How this dynamic equilibrium is achieved, in particular following the clonal expansion and subsequent contraction of different cells after infection, remains poorly understood. Expression of the proteasome immunosubunits has been shown to influence not only major histocompatibility complex class I (MHC-I) antigen processing and thereby T-cell responses, but also the CD4/CD8 T-cell ratios in lymphoid organs. We examined the relationships between these different immunosubunit-mediated effects in mice of various proteasome subunit compositions during infection with Listeria monocytogenes. Mice that lacked the immunosubunit multicatalytic endopeptidase complex-like 1 (MECL-1) maintained enhanced CD4/CD8 T-cell ratios during infection, while MHC-I surface levels resembled those in wild-type (wt) mice. LMP7 gene-deficient mice, on the other hand, showed reduced MHC-I expression, while their splenic CD4/CD8 ratios were similar to those in wt mice. Remarkably, analysis of bone marrow-chimeric immunosubunit gene-deficient mice, reconstituted with a mixture of wt and LMP7- plus MECL-1-deficient bone marrow, revealed that the LMP7- plus MECL-1-deficient T-cell population maintained a higher CD4/CD8 T-cell ratio than the wt T-cell population before, during, and after infection and T-cell memory formation. Since in these mice the immunosubunit-positive and immunosubunit-negative T-cell populations were selected in the same thymus and expanded in the same lymphoid environments, our findings indicate that MECL-1 influences the homeostatic equilibrium between T-cell subsets, not through indirect extracellular signals, such as MHC-I expression or the cytokine milieu, but through direct effects on T-cell-intrinsic processes.

FRG1P-mediated aggregation of proteins involved in pre-mRNA processing.

FRG1 is considered a candidate gene for facioscapulohumeral muscular dystrophy (FSHD) based on its location at chromosome 4qter and its upregulation in FSHD muscle. The FRG1 protein (FRG1P) localizes to nucleoli, Cajal bodies (and speckles), and has been suggested to be a component of the human spliceosome but its exact function is unknown. Recently, transgenic mice overexpressing high levels of FRG1P in skeletal muscle were described to present with muscular dystrophy. Moreover, upregulation of FRG1P was demonstrated to correlate with missplicing of specific pre-mRNAs. In this study, we have combined colocalization studies with yeast two-hybrid screens to identify proteins that associate with FRG1P. We demonstrate that artificially induced nucleolar aggregates of VSV-FRG1P specifically sequester proteins involved in pre-mRNA processing. In addition, we have identified SMN, PABPN1, and FAM71B, a novel speckle and Cajal body protein, as binding partners of FRG1P. All these proteins are, or seem to be, involved in RNA biogenesis. Our data confirm the presence of FRG1P in protein complexes containing human spliceosomes and support a potential role of FRG1P in either splicing or another step in nuclear RNA biogenesis. Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes.