CD4+ T Cells From Individuals With Type 1 Diabetes Respond to a Novel Class of Deamidated Peptides Formed in Pancreatic Islets.

The ß-cell plays a crucial role in the pathogenesis of type 1 diabetes, in part through the posttranslational modification of self-proteins by biochemical processes such as deamidation. These neoantigens are potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 disease-related proteins within inflammatory cytokine-stressed human islets of Langerhans. T-cell clones responsive against one Gln- and three Asn-deamidated peptides could be isolated from peripheral blood of individuals with type 1 diabetes. Ex vivo HLA class II tetramer staining detected higher T-cell frequencies in individuals with the disease compared with control individuals. Furthermore, there was a positive correlation between the frequencies of T cells specific for deamidated peptides, insulin antibody levels at diagnosis, and duration of disease. These results highlight that stressed human islets are prone to enzymatic and biochemical deamidation and suggest that both Gln- and Asn-deamidated peptides can promote the activation and expansion of autoreactive CD4+ T cells. These findings add to the growing evidence that posttranslational modifications undermine tolerance and may open the road for the development of new diagnostic and therapeutic applications for individuals living with type 1 diabetes.

Redox regulation of m6A methyltransferase METTL3 in ß-cells controls the innate immune response in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by the destruction of pancreatic ß-cells. Several observations have renewed the interest in ß-cell RNA sensors and editors. Here, we report that N6-methyladenosine (m6A) is an adaptive ß-cell safeguard mechanism that controls the amplitude and duration of the antiviral innate immune response at T1D onset. m6A writer methyltransferase 3 (METTL3) levels increase drastically in ß-cells at T1D onset but rapidly decline with disease progression. m6A sequencing revealed the m6A hypermethylation of several key innate immune mediators, including OAS1, OAS2, OAS3 and ADAR1 in human islets and EndoC-ßH1 cells at T1D onset. METTL3 silencing enhanced 2'-5'-oligoadenylate synthetase levels by increasing its mRNA stability. Consistently, in vivo gene therapy to prolong Mettl3 overexpression specifically in ß-cells delayed diabetes progression in the non-obese diabetic mouse model of T1D. Mechanistically, the accumulation of reactive oxygen species blocked upregulation of METTL3 in response to cytokines, while physiological levels of nitric oxide enhanced METTL3 levels and activity. Furthermore, we report that the cysteines in position C276 and C326 in the zinc finger domains of the METTL3 protein are sensitive to S-nitrosylation and are important to the METTL3-mediated regulation of oligoadenylate synthase mRNA stability in human ß-cells. Collectively, we report that m6A regulates the innate immune response at the ß-cell level during the onset of T1D in humans.

Interferons are key cytokines acting on pancreatic islets in type 1 diabetes.

AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.

Protection of ß cells against pro-inflammatory cytokine stress by the GDF15-ERBB2 signaling.

Aim/hypothesis: Growth/differentiation factor 15 (GDF15) is a therapeutic target for a variety of metabolic diseases, including type 1 diabetes (T1D). However, the nausea caused by GDF15 is a challenging point for therapeutic development. In addition, it is unknown why the endogenous GDF15 fails to protect from T1D development. Here, we investigate the GDF15 signaling in pancreatic islets towards opening possibilities for therapeutic targeting in ß cells and to understand why this protection fails to occur naturally. Methods: GDF15 signaling in islets was determined by proximity-ligation assay, untargeted proteomics, pathway analysis, and treatment of cells with specific inhibitors. To determine if GDF15 levels would increase prior to disease onset, plasma levels of GDF15 were measured in a longitudinal prospective study of children during T1D development (n=132 cases vs. n=40 controls) and in children with islet autoimmunity but normoglycemia (n=47 cases vs. n=40 controls) using targeted mass spectrometry. We also investigated the regulation of GDF15 production in islets by fluorescence microscopy and western blot analysis. Results: The proximity-ligation assay identified ERBB2 as the GDF15 receptor in islets, which was confirmed using its specific antagonist, tucatinib. The untargeted proteomics analysis and caspase assay showed that ERBB2 activation by GDF15 reduces ß cell apoptosis by downregulating caspase 8. In plasma, GDF15 levels were higher (p=0.0024) during T1D development compared to controls, but not in islet autoimmunity with normoglycemia. However, in the pancreatic islets GDF15 was depleted via sequestration of its mRNA into stress granules, resulting in translation halting. Conclusions/interpretation: GDF15 protects against T1D via ERBB2-mediated decrease of caspase 8 expression in pancreatic islets. Circulating levels of GDF15 increases pre-T1D onset, which is insufficient to promote protection due to its localized depletion in the islets. These findings open opportunities for targeting GDF15 downstream signaling for pancreatic ß cell protection in T1D and help to explain the lack of natural protection by the endogenous protein.

Stress-induced ß cell early senescence confers protection against type 1 diabetes.

During the progression of type 1 diabetes (T1D), ß cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve ß cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in ß cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the ß cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced ß cells, the reduction of ß cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual ß cells of T1D patients. Our findings reveal a previously unrecognized link between ß cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.

Myo9b mutations are associated with altered dendritic cell functions and increased susceptibility to autoimmune diabetes onset.

The regulation of autoimmunity against pancreatic islet ß cells for type 1 diabetes (T1D) onset is still unclear. NOD/ShiLtJ (NOD) mice are prone to the onset of autoimmune diabetes, but its congenic strain, ALR/Lt (ALR), is not. Here we show that dendritic cells (DC) in ALR mice have impaired migratory and T-cell priming capability. Genomic comparative analysis maps a 33-bp deletion in the ALR Myosin IXb (Myo9b) gene when compared with NOD genome; meanwhile, data from knock-in models show that this ALR Myo9b allele impairs phenotypic and functional maturation of DCs, and prevents the development and progression of spontaneous autoimmune diabetes in NOD mice. In parallel, while the ALR 33-bp deletion of Myo9b is not conserved in human, we find a MYO9B R133Q polymorphism associating with increased risk of T1D and enhanced DC function in patients with T1D. Our results thus hint that alterations in Myo9b may contribute to altered DC function and autoimmune diabetes onset.

Adipose tissue plasticity in pheochromocytoma patients suggests a role of the splicing machinery in human adipose browning.

Adipose tissue from pheochromocytoma patients acquires brown fat features, making it a valuable model for studying the mechanisms that control thermogenic adipose plasticity in humans. Transcriptomic analyses revealed a massive downregulation of splicing machinery components and splicing regulatory factors in browned adipose tissue from patients, with upregulation of a few genes encoding RNA-binding proteins potentially involved in splicing regulation. These changes were also observed in cell culture models of human brown adipocyte differentiation, confirming a potential involvement of splicing in the cell-autonomous control of adipose browning. The coordinated changes in splicing are associated with a profound modification in the expression levels of splicing-driven transcript isoforms for genes involved in the specialized metabolism of brown adipocytes and those encoding master transcriptional regulators of adipose browning. Splicing control appears to be a relevant component of the coordinated gene expression changes that allow human adipose tissue to acquire a brown phenotype.

BCL-XL Overexpression Protects Pancreatic ß-Cells against Cytokine- and Palmitate-Induced Apoptosis.

Diabetes is a chronic disease that affects glucose metabolism, either by autoimmune-driven ß-cell loss or by the progressive loss of ß-cell function, due to continued metabolic stresses. Although both α- and ß-cells are exposed to the same stressors, such as proinflammatory cytokines and saturated free fatty acids (e.g., palmitate), only α-cells survive. We previously reported that the abundant expression of BCL-XL, an anti-apoptotic member of the BCL-2 family of proteins, is part of the α-cell defense mechanism against palmitate-induced cell death. Here, we investigated whether BCL-XL overexpression could protect ß-cells against the apoptosis induced by proinflammatory and metabolic insults. For this purpose, BCL-XL was overexpressed in two ß-cell lines-namely, rat insulinoma-derived INS-1E and human insulin-producing EndoC-ßH1 cells-using adenoviral vectors. We observed that the BCL-XL overexpression in INS-1E cells was slightly reduced in intracellular Ca2+ responses and glucose-stimulated insulin secretion, whereas these effects were not observed in the human EndoC-ßH1 cells. In INS-1E cells, BCL-XL overexpression partially decreased cytokine- and palmitate-induced ß-cell apoptosis (around 40% protection). On the other hand, the overexpression of BCL-XL markedly protected EndoC-ßH1 cells against the apoptosis triggered by these insults (>80% protection). Analysis of the expression of endoplasmic reticulum (ER) stress markers suggests that resistance to the cytokine and palmitate conferred by BCL-XL overexpression might be, at least in part, due to the alleviation of ER stress. Altogether, our data indicate that BCL-XL plays a dual role in ß-cells, participating both in cellular processes related to ß-cell physiology and in fostering survival against pro-apoptotic insults.

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates TNF-α-induced human beta cell dysfunction and death.

AIMS/HYPOTHESIS: TNF-α plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-α inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-α, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. METHODS: EndoC-ßH1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-α (2000 U/ml) or TNF-α (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. RESULTS: PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-α and/or TNF-α by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-α, TNF-α or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-α-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-α cytotoxicity. CONCLUSIONS/INTERPRETATION: We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-α in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-α.

Redox Regulation of m 6 A Methyltransferase METTL3 in Human ß-cells Controls the Innate Immune Response in Type 1 Diabetes.

Type 1 Diabetes (T1D) is characterized by autoimmune-mediated destruction of insulin-producing ß-cells. Several observations have renewed interest in the innate immune system as an initiator of the disease process against ß-cells. Here, we show that N 6 -Methyladenosine (m 6 A) is an adaptive ß-cell safeguard mechanism that accelerates mRNA decay of the 2'-5'-oligoadenylate synthetase (OAS) genes to control the antiviral innate immune response at T1D onset. m 6 A writer methyltransferase 3 (METTL3) levels increase drastically in human and mouse ß-cells at T1D onset but rapidly decline with disease progression. Treatment of human islets and EndoC-ßH1 cells with pro-inflammatory cytokines interleukin-1 ß and interferon α mimicked the METTL3 upregulation seen at T1D onset. Furthermore, m 6 A-sequencing revealed the m 6 A hypermethylation of several key innate immune mediators including OAS1, OAS2, and OAS3 in human islets and EndoC-ßH1 cells challenged with cytokines. METTL3 silencing in human pseudoislets or EndoC-ßH1 cells enhanced OAS levels by increasing its mRNA stability upon cytokine challenge. Consistently, in vivo gene therapy, to prolong Mettl3 overexpression specifically in ß-cells, delayed diabetes progression in the non-obese diabetic (NOD) mouse model of T1D by limiting the upregulation of Oas pointing to potential therapeutic relevance. Mechanistically, the accumulation of reactive oxygen species blocked METTL3 upregulation in response to cytokines, while physiological levels of nitric oxide promoted its expression in human islets. Furthermore, for the first time to our knowledge, we show that the cysteines in position C276 and C326 in the zinc finger domain of the METTL3 protein are sensitive to S-nitrosylation (SNO) and are significant for the METTL3 mediated regulation of OAS mRNA stability in human ß-cells in response to cytokines. Collectively, we report that m 6 A regulates human and mouse ß-cells to control the innate immune response during the onset of T1D and propose targeting METTL3 to prevent ß-cell death in T1D.

Recognition of mRNA Splice Variant and Secretory Granule Epitopes by CD4+ T Cells in Type 1 Diabetes.

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.

The type 1 diabetes gene TYK2 regulates ß-cell development and its responses to interferon-α.

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic ß-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in ß-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8+ T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in ß-cell development and support TYK2 inhibition in adult ß-cells as a potent therapeutic target to halt T1D progression.

The Role of Beta Cell Recovery in Type 2 Diabetes Remission.

Type 2 diabetes (T2D) has been considered a relentlessly worsening disease, due to the progressive deterioration of the pancreatic beta cell functional mass. Recent evidence indicates, however, that remission of T2D may occur in variable proportions of patients after specific treatments that are associated with recovery of beta cell function. Here we review the available information on the recovery of beta cells in (a) non-diabetic individuals previously exposed to metabolic stress; (b) T2D patients following low-calorie diets, pharmacological therapies or bariatric surgery; (c) human islets isolated from non-diabetic organ donors that recover from "lipo-glucotoxic" conditions; and (d) human islets isolated from T2D organ donors and exposed to specific treatments. The improvement of insulin secretion reported by these studies and the associated molecular traits unveil the possibility to promote T2D remission by directly targeting pancreatic beta cells.

A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes.

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.

Kdm2a deficiency in macrophages enhances thermogenesis to protect mice against HFD-induced obesity by enhancing H3K36me2 at the Pparg locus.

Kdm2a catalyzes H3K36me2 demethylation to play an intriguing epigenetic regulatory role in cell proliferation, differentiation, and apoptosis. Herein we found that myeloid-specific knockout of Kdm2a (LysM-Cre-Kdm2af/f, Kdm2a-/-) promoted macrophage M2 program by reprograming metabolic homeostasis through enhancing fatty acid uptake and lipolysis. Kdm2a-/- increased H3K36me2 levels at the Pparg locus along with augmented chromatin accessibility and Stat6 recruitment, which rendered macrophages with preferential M2 polarization. Therefore, the Kdm2a-/- mice were highly protected from high-fat diet (HFD)-induced obesity, insulin resistance, and hepatic steatosis, and featured by the reduced accumulation of adipose tissue macrophages and repressed chronic inflammation following HFD challenge. Particularly, Kdm2a-/- macrophages provided a microenvironment in favor of thermogenesis. Upon HFD or cold challenge, the Kdm2a-/- mice manifested higher capacity for inducing adipose browning and beiging to promote energy expenditure. Collectively, our findings demonstrate the importance of Kdm2a-mediated H3K36 demethylation in orchestrating macrophage polarization, providing novel insight that targeting Kdm2a in macrophages could be a viable therapeutic approach against obesity and insulin resistance.

Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

A nanobody-based nuclear imaging tracer targeting dipeptidyl peptidase 6 to determine the mass of human beta cell grafts in mice.

AIMS/HYPOTHESIS: Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody '4hD29') against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). METHODS: DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-ßH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-ßH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. RESULTS: The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-ßH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-ßH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. CONCLUSIONS/INTERPRETATION: These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.

Loss of ubiquitin-conjugating enzyme E2 (Ubc9) in macrophages exacerbates multiple low-dose streptozotocin-induced diabetes by attenuating M2 macrophage polarization.

Type 1 diabetes (T1D) is characterized by the selective autoimmune destruction of the islet ß cells, and macrophages play a significant role in this process. Small ubiquitin-like modification (SUMOylation) is an important posttranslational modification involved in T1D pathogenesis, but its function in macrophages remains unexplored. We presently developed and used macrophage-specific ubiquitin-conjugating enzyme E2 (Ubc9) knockout (LyzM-Cre-Ubc9fl/fl, KO) mice to address the impact of SUMOylation on macrophage function in a T1D model. We observed that blocking Ubc9 in macrophages exacerbated multiple-low dose streptozotocin (MLD-STZ)-induced diabetes. Specifically, after STZ treatment, blood glucose levels were consistently elevated in the KO mice. The KO mice exhibited a higher diabetes incidence than WT controls (85% vs. 55%, P < 0.01) along with a higher insulitis severity. The loss of Ubc9 impaired macrophage energy metabolism and attenuated macrophage M2 program, thereby enhancing T cell activation. Pancreas-resident macrophages, rather than migrant macrophages, played a predominant role in MLD-STZ-induced diabetes. Mechanistically, Ubc9-mediated SUMOylation of interferon regulator factor 4 (IRF4) enhanced its nuclear localization and stability, thereby transcribing IL-4 and arginase 1 (Arg1) to promote the macrophage M2 program. Ubc9-mediated SUMOylation modulates T1D risk at least in part by regulating macrophage function. Modulation of disturbed SUMOylation process in macrophages, either through cell adoptive transfer or targeted drug-delivery, could help to establish a tolerant pancreatic microenvironment and promote inflammation resolution in early insulitis stage, thus hindering T1D progression.

Cytokine-induced translocation of GRP78 to the plasma membrane triggers a pro-apoptotic feedback loop in pancreatic beta cells.

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-ßH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.

MCPIP1 regulates the sensitivity of pancreatic beta-cells to cytokine toxicity.

The autoimmune-mediated beta-cell death in type 1 diabetes (T1DM) is associated with local inflammation (insulitis). We examined the role of MCPIP1 (monocyte chemotactic protein-induced protein 1), a novel cytokine-induced antiinflammatory protein, in this process. Basal MCPIP1 expression was lower in rat vs. human islets and beta-cells. Proinflammatory cytokines stimulated MCPIP1 expression in rat and human islets and in insulin-secreting cells. Moderate overexpression of MCPIP1 protected insulin-secreting INS1E cells against cytokine toxicity by a mechanism dependent on the presence of the PIN/DUB domain in MCPIP1. It also reduced cytokine-induced Chop and C/ebpß expression and maintained MCL-1 expression. The shRNA-mediated suppression of MCPIP1 led to the potentiation of cytokine-mediated NFκB activation and cytokine toxicity in human EndoC-ßH1 beta-cells. MCPIP1 expression was very high in infiltrated beta-cells before and after diabetes manifestation in the LEW.1AR1-iddm rat model of human T1DM. The extremely high expression of MCPIP1 in clonal beta-cells was associated with a failure of the regulatory feedback-loop mechanism, ER stress induction and high cytokine toxicity. In conclusion, our data indicate that the expression level of MCPIP1 affects the susceptibility of insulin-secreting cells to cytokines and regulates the mechanism of beta-cell death in T1DM.