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.

Exercise as a non-pharmacological intervention to protect pancreatic beta cells in individuals with type 1 and type 2 diabetes.

AIMS/HYPOTHESIS: Diabetes is characterised by progressive loss of functional pancreatic beta cells. None of the therapeutic agents used to treat diabetes arrest this process; preventing beta cell loss remains a major unmet need. We have previously shown that serum from eight young healthy male participants who exercised for 8 weeks protected human islets and insulin-producing EndoC-ßH1 cells from apoptosis induced by proinflammatory cytokines or the endoplasmic reticulum (ER) stressor thapsigargin. Whether this protective effect is influenced by sex, age, training modality, ancestry or diabetes is unknown. METHODS: We enrolled 82 individuals, male or female, non-diabetic or diabetic, from different origins, in different supervised training protocols for 8-12 weeks (including training at home during the COVID-19 pandemic). EndoC-ßH1 cells were treated with 'exercised' serum or with the exerkine clusterin to ascertain cytoprotection from ER stress. RESULTS: The exercise interventions were effective and improved [Formula: see text] values in both younger and older, non-obese and obese, non-diabetic and diabetic participants. Serum obtained after training conferred significant beta cell protection (28% to 35% protection after 4 and 8 weeks of training, respectively) from severe ER stress-induced apoptosis. Cytoprotection was not affected by the type of exercise training or participant age, sex, BMI or ancestry, and persisted for up to 2 months after the end of the training programme. Serum from exercised participants with type 1 or type 2 diabetes was similarly protective. Clusterin reproduced the beneficial effects of exercised sera. CONCLUSIONS/INTERPRETATION: These data uncover the unexpected potential to preserve beta cell health by exercise training, opening a new avenue to prevent or slow diabetes progression through humoral muscle-beta cell crosstalk.

Preclinical evaluation of tyrosine kinase 2 inhibitors for human beta-cell protection in type 1 diabetes.

AIM: Type 1 diabetes (T1D) is a chronic autoimmune disease leading to progressive loss of pancreatic beta cells. Interferon (IFN)-α plays a critical role in the crosstalk between pancreatic beta cells and the immune system in early insulitis. In human beta cells IFNα signals through JAK1 and TYK2, leading to endoplasmic reticulum stress, inflammation and HLA class I overexpression. IFNα, acting synergistically with IL-1ß, induces apoptosis. Polymorphisms in TYK2 that decrease its activity are associated with protection against T1D, and we hypothesized that pharmacological inhibitors that specifically target TYK2 could protect human beta cells against the deleterious effects of IFNα. MATERIALS AND METHODS: Two TYK2 inhibitors provided by Nimbus Lakshmi were tested in human insulin-producing EndoC-ßH1 cells and human islets to evaluate their effect on IFNα signalling, beta-cell function and susceptibility to viral infection using RT-qPCR, western blot, immunofluorescence, ELISA and nuclear dyes. RESULTS: The two TYK2 inhibitors tested prevented IFNα-induced human beta-cell gene expression in a dose-dependent manner. They also protected human islets against IFNα + IL-1ß-induced apoptosis. Importantly, these inhibitors did not modify beta-cell function or their survival following infection with the potential diabetogenic coxsackieviruses CVB1 and CVB5. CONCLUSIONS: The two TYK2 inhibitors tested inhibit the IFNα signalling pathway in human beta cells, decreasing its pro-inflammatory and pro-apoptotic effects without sensitizing the cells to viral infection. The preclinical findings could pave the way for future clinical trials with TYK2 inhibitors for the prevention and treatment of type 1 diabetes.

IFN-α induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells.

AIMS/HYPOTHESIS: IFN-α, a cytokine expressed in human islets from individuals affected by type 1 diabetes, plays a key role in the pathogenesis of diabetes by upregulating inflammation, endoplasmic reticulum (ER) stress and MHC class I overexpression, three hallmarks of islet histology in early type 1 diabetes. We tested whether expression of these mediators of beta cell loss is reversible upon IFN-α withdrawal or IFN-α pathway inhibition. METHODS: IFN-α-induced MHC class I overexpression, ER stress and inflammation were evaluated by flow cytometry, immunofluorescence and real-time PCR in human EndoC-ßH1 cells or human islets exposed to IFN-α with or without the presence of Janus kinase (JAK) inhibitors. Protein expression was evaluated by western blot. RESULTS: IFN-α-induced expression of inflammatory and ER stress markers returned to baseline after 24-48 h following cytokine removal. In contrast, MHC class I overexpression at the cell surface persisted for at least 7 days. Treatment with JAK inhibitors, when added with IFN-α, prevented MHC class I overexpression, but when added 24 h after IFN-α exposure these inhibitors failed to accelerate MHC class I return to baseline. CONCLUSIONS/INTERPRETATION: IFN-α mediates a long-lasting and preferential MHC class I overexpression in human beta cells, which is not affected by the subsequent addition of JAK inhibitors. These observations suggest that IFN-α-stimulated long-lasting MHC class I expression may amplify beta cell antigen presentation during the early phase of type 1 diabetes and that IFN-α inhibitors might need to be used at very early stages of the disease to be effective.

Interferon-α mediates human beta cell HLA class I overexpression, endoplasmic reticulum stress and apoptosis, three hallmarks of early human type 1 diabetes.

AIMS/HYPOTHESIS: Three hallmarks of the pancreatic islets in early human type 1 diabetes are overexpression of HLA class I, endoplasmic reticulum (ER) stress and beta cell apoptosis. The mediators of these phenomena remain to be defined. The type I interferon IFNα is expressed in human islets from type 1 diabetes patients and mediates HLA class I overexpression. We presently evaluated the mechanisms involved in IFNα-induced HLA class I expression in human beta cells and determined whether this cytokine contributes to ER stress and apoptosis. METHODS: IFNα-induced inflammation, ER stress and apoptosis were evaluated by RT-PCR, western blot, immunofluorescence and nuclear dyes, and proteins involved in type I interferon signalling were inhibited by small interfering RNAs. All experiments were performed in human islets or human EndoC-ßH1 cells. RESULTS: IFNα upregulates HLA class I, inflammation and ER stress markers in human beta cells via activation of the candidate gene TYK2, and the transcription factors signal transducer and activator of transcription 2 and IFN regulatory factor 9. Furthermore, it acts synergistically with IL-1ß to induce beta cell apoptosis. CONCLUSIONS/INTERPRETATION: The innate immune effects induced by IFNα may induce and amplify the adaptive immune response against human beta cells, indicating that IFNα has a central role in the early phases of diabetes.

FOXO transcription factors in cancer development and therapy.

The forkhead box O (FOXO) transcription factors are considered as tumor suppressors that limit cell proliferation and induce apoptosis. FOXO gene alterations have been described in a limited number of human cancers, such as rhabdomyosarcoma, leukemia and lymphoma. In addition, FOXO proteins are inactivated by major oncogenic signals such as the phosphatidylinositol-3 kinase pathway and MAP kinases. Their expression is also repressed by micro-RNAs in multiple cancer types. FOXOs are mediators of the tumor response to various therapies. However, paradoxical roles of FOXOs in cancer progression were recently described. FOXOs contribute to the maintenance of leukemia-initiating cells in acute and chronic myeloid leukemia. These factors may also promote invasion and metastasis of subsets of colon and breast cancers. Resistance to treatment was also ascribed to FOXO activation in multiple cases, including targeted therapies. In this review, we discuss the complex role of FOXOs in cancer development and response to therapy.

The expression of the tumour suppressor HBP1 is down-regulated by growth factors via the PI3K/PKB/FOXO pathway.

Growth factors inactivate the FOXO (forkhead box O) transcription factors through PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B). By comparing microarray data from multiple model systems, we identified HBP1 (high-mobility group-box protein 1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), PI3K and PKB, whereas it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukaemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and ChIP, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by shRNA increased the proliferation of human fibroblasts in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analysing a transcriptomics dataset from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumours that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K/FOXO pathway and controls cell proliferation in response to growth factors.

Pharmacological inhibition of tyrosine protein-kinase 2 reduces islet inflammation and delays type 1 diabetes onset in mice.

Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human ß cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced ß cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of ß cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented ß cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for ß cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.

Endogenous mitochondrial double-stranded RNA is not an activator of the type I interferon response in human pancreatic beta cells.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells. Interferon-α (IFNα), an antiviral cytokine, is expressed in the pancreatic islets in early T1D, which may be secondary to viral infections. However, not all patients harboring a type I IFN signature present signals of viral infection, suggesting that this response might be initiated by other "danger signals". Accumulation of mitochondrial double-stranded RNA (mtdsRNA; a danger signal), secondary to silencing of members of the mitochondrial degradosome, PNPT1 and SUV3, has been described to activate the innate immune response. METHODS: To evaluate whether mtdsRNA represents a "danger signal" for pancreatic beta cells in the context of T1D, we silenced PNPT1 and/or SUV3 in slowly proliferating human insulin-secreting EndoC-ßH1 cells and in non-proliferating primary human beta cells and evaluated dsRNA accumulation by immunofluorescence and the type I IFN response by western blotting and RT-qPCR. RESULTS: Only the simultaneous silencing of PNPT1/SUV3 induced dsRNA accumulation in EndoC-ßH1 cells but not in dispersed human islets, and there was no induction of a type I IFN response. By contrast, silencing of these two genes individually was enough to induce dsRNA accumulation in fibroblasts present in the human islet preparations. CONCLUSIONS: These data suggest that accumulation of endogenous mtdsRNA following degradosome knockdown depends on the proliferative capacity of the cells and is not a mediator of the type I IFN response in human pancreatic beta cells.

An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells.

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.

PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.

BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.

PDGF-induced fibroblast growth requires monounsaturated fatty acid production by stearoyl-CoA desaturase.

Stearoyl-coenzyme A desaturase (SCD) catalyzes the Δ9-cis desaturation of saturated fatty acids (SFA) to generate monounsaturated fatty acids (MUFA). This enzyme is highly up-regulated by platelet-derived growth factor (PDGF) in human fibroblasts. Accordingly, the analysis of cellular fatty acids by gas chromatography showed that PDGF significantly increased the proportion of MUFA, particularly palmitoleate, in cellular lipids. To further analyze the role of SCD in fibroblasts, we used small hairpin RNA targeting SCD (shSCD), which decreased the MUFA content. SCD down-regulation blunted the proliferation of fibroblasts in response to PDGF. This was confirmed using a pharmacological inhibitor of SCD. In addition, proliferation was blocked by palmitate and stearate (two SCD substrates) but not by palmitoleate and oleate (two SCD products). In the presence of an equal amount of oleate, palmitate had no effect on cell proliferation. SCD inhibition or down-regulation did not decrease PDGF receptor activity or signaling. However, by measuring plasma membrane lipid lateral diffusion by fluorescence recovery after photobleaching, we showed that the modulation of the MUFA/SFA ratio by PDGF and SCD inhibitor was related to modifications of membrane fluidity. Altogether, our data suggest that SCD is required for the response of normal fibroblasts to growth factors.

PDGF-D expression is down-regulated by TGFß in fibroblasts.

Transforming growth factor-ß (TGFß) is a key mediator of fibrogenesis. TGFß is overexpressed and activated in fibrotic diseases, regulates fibroblast differentiation into myofibroblasts and induces extracellular matrix deposition. Platelet-derived growth factor (PDGF) is also a regulator of fibrogenesis. Some studies showed a link between TGFß and PDGF in certain fibrotic diseases. TGFß induces PDGF receptor alpha expression in scleroderma fibroblasts. PDGF-C and -D are the most recently discovered ligands and also play a role in fibrosis. In this study, we report the first link between TGFß and PDGF-D and -C ligands. In normal fibroblasts, TGFß down-regulated PDGF-D expression and up-regulated PDGF-C expression at the mRNA and protein levels. This phenomenon is not limited to TGFß since other growth factors implicated in fibrosis, such as FGF, EGF and PDGF-B, also regulated PDGF-D and PDGF-C expression. Among different kinase inhibitors, only TGFß receptor inhibitors and the IκB kinase (IKK) inhibitor BMS-345541 blocked the effect of TGFß. However, activation of the classical NF-κB pathway was not involved. Interestingly, in a model of lung fibrosis induced by either bleomycin or silica, PDGF-D was down-regulated, which correlates with the production of TGFß and other fibrotic growth factors. In conclusion, the down-regulation of PDGF-D by TGFß and other growth factors may serve as a negative feedback in the network of cytokines that control fibrosis.