Upregulation of HLA class II in pancreatic beta cells from organ donors with type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to characterise and quantify the expression of HLA class II (HLA-II) in human pancreatic tissue sections and to analyse its induction in human islets. METHODS: We immunostained human pancreatic tissue sections from non-diabetic (n = 5), autoantibody positive (Aab+; n = 5), and type 1 diabetic (n = 5) donors, obtained from the Network of Pancreatic Organ Donors (nPOD), with HLA-II, CD68 and insulin. Each tissue section was acquired with a widefield slide scanner and then analysed with QuPath software. In total, we analysed 7415 islets that contained 338,480 cells. Widefield microscopy was further complemented by high resolution imaging of 301 randomly selected islets, acquired using a Zeiss laser scanning confocal (LSM880) to confirm our findings. Selected beta cells were acquired in enhanced resolution using LSM880 with an Airyscan detector. Further, we cultured healthy isolated human islets and reaggregated human islet microtissues with varying concentrations of proinflammatory cytokines (IFN-γ, TNF-α and IL-1ß). After proinflammatory cytokine culture, islet function was measured by glucose-stimulated insulin secretion, and HLA-I and HLA-II expression was subsequently evaluated with immunostaining or RNA sequencing. RESULTS: Insulin-containing islets (ICIs) of donors with type 1 diabetes had a higher percentage of HLA-II positive area (24.31%) compared with type 1 diabetic insulin-deficient islets (IDIs, 0.67%), non-diabetic (3.80%), and Aab+ (2.31%) donors. In ICIs of type 1 diabetic donors, 45.89% of the total insulin signal co-localised with HLA-II, and 27.65% of the islet beta cells expressed both HLA-II and insulin, while in non-diabetic and Aab+ donors 0.96% and 0.59% of the islet beta cells, respectively, expressed both markers. In the beta cells of donors with type 1 diabetes, HLA-II was mostly present in the cell cytoplasm, co-localising with insulin. In the experiments with human isolated islets and reaggregated human islets, we observed changes in insulin secretion upon stimulation with proinflammatory cytokines, as well as higher expression of HLA-II and HLA-I when compared with controls cultured with media, and an upregulation of HLA-I and HLA-II RNA transcripts. CONCLUSIONS/INTERPRETATION: After a long-standing controversy, we provide definitive evidence that HLA-II can be expressed by pancreatic beta cells from patients with type 1 diabetes. Furthermore, this upregulation can be induced in vitro in healthy isolated human islets or reaggregated human islets by treatment with proinflammatory cytokines. Our findings support a role for HLA-II in type 1 diabetes pathogenesis since HLA-II expressing beta cells can potentially become a direct target of autoreactive CD4+ lymphocytes.

HLA class I hyper-expression unmasks beta cells but not alpha cells to the immune system in pre-diabetes.

Human leukocyte antigens of class-I (HLA-I) molecules are hyper-expressed in insulin-containing islets (ICI) of type 1 diabetic (T1D) donors. This study investigated the HLA-I expression in autoantibody positive (AAB+) donors and defined its intra-islet and intracellular localization as well as proximity to infiltrating CD8 T cells with high-resolution confocal microscopy. We found HLA-I hyper-expression had already occurred prior to clinical diagnosis of T1D in islets of AAB+ donors. Interestingly, throughout all stages of disease, HLA-I was mostly expressed by alpha cells. Hyper-expression in AAB+ and T1D donors was associated with intra-cellular accumulation in the Golgi. Proximity analysis showed a moderate but significant correlation between HLA-I and infiltrating CD8 T cells only in ICI of T1D donors, but not in AAB+ donors. These observations not only demonstrate a very early, islet-intrinsic immune-independent increase of HLA-I during diabetes pathogenesis, but also point towards a role for alpha cells in T1D.

IL-17 is expressed on beta and alpha cells of donors with type 1 and type 2 diabetes.

PURPOSE: IL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. METHODS: We stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. RESULTS: We observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. CONCLUSION: Our finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.

Human herpesvirus-6 is present at higher levels in the pancreatic tissues of donors with type 1 diabetes.

Human herpesvirus-6 (HHV-6) is a ubiquitous pathogen associated with nervous and endocrine autoimmune disorders. The aim of this study was to investigate the presence of HHV-6 in pancreatic tissue sections from non-diabetic, auto-antibody positive (AAB+), and donors with type 1 diabetes (T1D) and explore whether there is any association between HHV-6 and MHC class I hyperexpression and CD8 T cell infiltration. HHV-6 DNA was detected by PCR and its protein was examined by indirect immunofluorescence assay followed by imaging using high-resolution confocal microscopy. Viral DNA (U67) was found in most pancreata of non-diabetic (3 out of 4), AAB+ (3 out of 5) and T1D donors (6 out of 7). Interestingly, HHV-6 glycoprotein B (gB) was more expressed in islets and exocrine pancreas of donors with T1D. However, gB expression was not directly associated with other pathologies. Out of 20 islets with high gB expression, only 3 islets (15%) showed MHC class I hyperexpression. Furthermore, no correlation was found between gB expression and CD8 T cell infiltration on a per-islet basis in any of the groups. Our observations indicate that HHV-6 DNA and protein are present in the pancreas of non-diabetic subjects but gB expression is higher in the pancreas of donors with T1D. The possible role of HHV-6 as a contributory factor for T1D should therefore be further investigated.

Exposure of Piglets to Enteroviruses Investigated by an Immunoassay Based on the EV-G1 VP4 Peptide.

OBJECTIVE: The aim of this study was to investigate the exposure of piglets to enteroviruses-G (EV-G) through the presence of antibodies in their serum. METHODS: Serum samples were obtained from the vena cava of 10 piglets at 9 weeks of age and again 39 days later (day 39). They were tested using an immunoassay based on the EV-G1 VP4 peptide, since VP4 is highly conserved among the four Enterovirus capsid proteins, and by using a seroneutralization assay. RESULTS: For each serum collected on day 39 the optical density was high compared to the value obtained in serum collected earlier (p = 0.002). However, the titers of anti-EV-G1 serum neutralizing activity were not different in paired samples (p > 0.999). The sequence alignment of the EV-G1 VP4 peptide, encompassing 50 amino acids, used in the immunoassay showed 88% homology with EV-G, suggesting that antibodies directed toward other EV-G than EV-G1 may be detected. CONCLUSION: An immunoassay based on EV-G1 VP4 can detect an increased level of EV-G antibodies in piglet serum samples. Further studies are needed to determine whether this immunoassay may be useful for diagnosis and/or epidemiological studies and to monitor EV-G infection in pigs to evaluate strategies aimed to prevent enterovirus infections.

Impact of coxsackievirus-B4E2 combined with a single low dose of streptozotocin on pancreas of outbred mice: investigation of viral load, pathology and inflammation.

Coxsackieviruses B (CV-B) belong to the EV-B species. CV-B and particularly CV-B4 are thought to be involved in the development of chronic diseases like type 1 diabetes (T1D). The mechanisms of the enteroviral pathogenesis of T1D are not well known, yet. The in vitro studies are rich with information but in vivo infection models are needed to investigate the impact of viruses onto organs. Our objective was to study the impact of CV-B4E2 combined with a single sub-diabetogenic dose of streptozotocin (STZ) on the pancreas of mice. The infection with CV-B4E2 of CD1 outbred mice treated with a sub-diabetogenic dose of STZ induced hyperglycemia and hypoinsulinemia. Along with the chemokine IP-10, viral RNA and infectious particles were detected in the pancreas. The pancreas of these animals was also marked with insulitis and other histological alterations. The model combining STZ and CV-B4E2 opens the door to new perspectives to better understand the interactions between virus and host, and the role of environmental factors capable, like STZ, to predispose the host to the diabetogenic effects of enteroviruses.

Fluoxetine can inhibit coxsackievirus-B4 E2 in vitro and in vivo.

Group B Coxsackieviruses (CV-B) are responsible for various acute human diseases, and they are involved in chronic diseases such as type 1 diabetes. It has been reported that fluoxetine (FLX) inhibited CV-B4E2 in human cell lines in vitro. In so far as CV-B4E2 can replicate in CD1 mice, it was investigated whether FLX could inhibit CV-B4E2 in vitro and in vivo in mouse systems. When 5.5 µM FLX was added to CV-B4E2-infected Min-6 cell (murine pancreas beta cell line) cultures, the virus-induced cytopathic effect was inhibited. In this system and in CV-B4E2-infected CD1 mouse pancreatic organotypic cultures treated with FLX the levels of infectious particles in supernatant fluids were below the limit of detection of the assay. The administration of FLX (10 mg/kg/day) by intraperitoneal route resulted in significant reduced levels of infectious particles in heart and pancreas of mice inoculated with CV-B4E2 by the same route. In conclusion FLX can inhibit CV-B4 in vitro and in vivo in mouse systems, additional studies are needed to investigate further the potential value of FLX to combat CV-B4 infections and to treat CV-B4-induced diseases.