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Inactivation of protein tyrosine phosphatases enhances interferon signaling in pancreatic islets.
William J. Stanley1,2, Sara A. Litwak1, Hong Sheng Quah1,2, Sih Min Tan3, Thomas W. H. Kay1,2, Tony Tiganis4, Judy B. de Haan3, Helen E. Thomas1,2, Esteban N. Gurzov1,2 1
St Vincent’s Institute of Medical Research, Melbourne, Australia, 2Department of
Medicine, St. Vincent’s Hospital, The University of Melbourne, Melbourne, Australia, 3Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia, 4Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia
Address correspondence and reprint requests to: Dr Esteban N. Gurzov St Vincent’s Institute of Medical Research 9 Princes Street Fitzroy, VIC, 3065 Phone: 61-3-9288-2480 Fax: 61-3-9416-2676 Email:
[email protected]
Disclosure statement: The authors declare no conflict of interest Running Title: Protein tyrosine phosphatase oxidation promotes inflammation
1 Diabetes Publish Ahead of Print, published online March 2, 2015
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ABSTRACT
Type 1 diabetes (T1D) is the result of autoimmune assault against the insulinproducing pancreatic β-cells, where chronic local inflammation (insulitis) leads to βcell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T cell invasion and activation. Cytokine signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from non-obese diabetic (NOD) mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling when compared to NOD mice that do not have any insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and β-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis.
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INTRODUCTION
Type 1 diabetes (T1D) is caused by progressive loss of pancreatic β-cells due to an autoimmune
assault.
Local
inflammation
and
pro-inflammatory
cytokines,
particularly interferons (IFNs), play an important role in β-cell loss (1; 2). IFN-γ signal transduction involves activation of the tyrosine kinases JAK1 and JAK2 that phosphorylate STAT1. These then dimerize, translocate to the nucleus, and bind γactivated sites of a diverse array of genes. Blocking activation of this transcriptional pathway protects islets from immune destruction (3-5). These data indicate that excessive activation of JAK/STAT signaling in islets during the inflammatory process contributes to β-cell dysfunction and death. Cytokine signaling results in the upregulation and release of factors by β-cells such as chemokines that attract immune cells and amplify the inflammatory process.
Protein tyrosine phosphatases (PTPs) are a large superfamily of enzymes that dephosphorylate tyrosine phosphorylated proteins to oppose the actions of protein tyrosine kinases (6; 7). PTPs play an important role in the development of both forms of diabetes (8). The architecture and low thiol pKa of the Cys residue in the active site of PTPs renders these proteins highly susceptible to oxidation by reactive oxygen species (ROS) (6; 8). ROS-mediated oxidation of the PTP active site Cys inhibits PTP activity and prevents substrate binding. It was recently established that PTP oxidation occurs in vivo under physiological and pathological conditions such as inflammation (6; 8; 9).
The total serum antioxidant levels of pre-diabetic and T1D patients are reduced in comparison to age-matched controls (10; 11). Furthermore, ROS and oxidative stress
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have been linked to β-cell cytotoxicity and are believed to play a role in pathology (12). The mechanism, however, remains unclear. Here, we show that oxidative stress in pancreatic islets during insulitis results in the enhanced oxidation of PTPs. Moreover, inactivation of PTPs in human and rodent islets and β-cells leads to increased interferon signaling, chemokine production and cell death.
RESEARCH DESIGN AND METHODS
Mice
Mice were maintained at St. Vincent’s Institute, and experiments were approved by the institutional animal ethics committee. The list of mice is provided in Supplementary Table 1.
Immunohistochemistry & immunofluorescence
Mouse pancreata were frozen in optimal cutting temperature (OCT) embedding medium (Sakura Finetek, CA) or fixed with formalin and embedded in paraffin. Sections were incubated with mouse anti-pSTAT1 (BD biosciences, CA) and guinea pig anti-insulin (DAKO, Denmark) or rabbit anti-nitrotyrosine (Millipore, Billeria, MA) primary antibodies. The sections were then incubated with secondary Alexafluo-488 anti-mouse and Alexa-fluo-568 anti-guinea pig (both from Molecular Probes,
Life
technologies,
CA)
and
counterstained
with
DAPI
for
immunofluorescence or secondary biotinylated anti-rabbit (Vector labs, Burlingame, CA) for immunohistochemistry.
Cell culture and treatments
Human pancreata were obtained, with informed consent from next-of-kin, from heart-
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beating, brain-dead donors by the Australian Islet Transplant Consortium, and approved by the human ethics committees of the hospitals involved and the Australian Red Cross. Human islets were isolated as described previously (13). Islets were used from non-diabetic donors with an average age of 64±3 years. Mouse islets were isolated as described previously (14). The insulin producing MIN6 and NIT-1 cell lines were cultured in DMEM (Invitrogen, UK) supplemented with 10% fetal calf serum. Cytokine concentrations were selected based on previous time course and dose-response studies (15).
Real-time PCR
Analyses were performed with the ddCT method using β-actin as internal controls. Probes are provided in Supplementary Table 2.
PTP oxidation, immunoprecipitation & Western blotting
Total (reversible and irreversible) PTP oxidation was assessed essentially as described previously (9). Briefly, PTP oxidation results in two pools of PTPs: oxidized (PTPSOH; inactive) and reduced (PTP-S−; active). To detect oxidized PTPs in NOD mice, pancreas samples were alkylated with N-ethylmaleimide (NEM, Sigma), rendering active PTPs resistant to further modification, while oxidized PTPs remain unaffected. Excess NEM was removed by column filtration, and reversibly oxidized PTPs reduced with dithiothreitol (DTT, Sigma). Following a buffer exchange, the reduced PTPs (representing PTPs that initially were reversibly oxidized) were oxidized to sulfonic acid (PTP-SO3H) using pervanadate (Sigma). Then, “hyper-oxidized” PTPs were detected by immunoblotting with the PTPox antibody (R&D Systems, Minneapolis, MN). Immunoprecipitation was performed using PTPox antibody (R&D
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Systems) and RIPA buffer. Antibody/protein complexes were collected with Protein G PLUS-Agarose (Santa Cruz Biotechnology, CA), washed, and then boiled in sample buffer (Santa Cruz Biotechnology) to remove the antibody/protein complex. Equal amounts of proteins were resolved by 10% SDS-PAGE and immunoblotted with the antibodies indicated in Supplementary Table 3. All blots shown are representative for 2-4 independent experiments.
Cell viability
Human islet preparations were assessed for viability by simultaneously staining monodispersed islet cells with 5 µg/ml 7-aminoactinomycin D (7-AAD) for islet viability, 10 µM Newport green (NPG) for β-cells and 1 µM tetramethylrhodamineethyl-ester (TMRE) for apoptosis (Molecular Probes, Invitrogen, Grand Island, NY), and analyzing by flow cytometry following the method of Ichii H et al (16). Mouse islets were dispersed into single cells with trypsin. DNA fragmentation was analyzed by staining with propidium iodide as previously described (17). The percentage cell death of MIN6 cells was determined in at least 600 cells per experimental condition by inverted fluorescence microscopy after staining with the DNA dyes Hoechst33342 (10 µg/ml) and propidium iodide (5 µg/ml).
Statistical analysis
Data are means ± SEM of 3-5 independent experiments. Comparisons between groups were made by paired t test or by ANOVA followed by t test with the Bonferroni correction.
RESULTS
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PTPs are oxidized in pancreases from non-obese diabetic (NOD) mice
To directly evaluate the role of insulitis-induced PTP oxidation, we used non-transgenic NOD mice and transgenic NOD mice expressing proinsulin (PI) in major histocompatibility complex (MHC) class II bearing cells (NOD PI mice) (18). NOD PI mice do not develop insulitis due to immune tolerance to proinsulin, but otherwise have a normal, functional immune system. At 12 weeks of age, NOD mice developed insulitis and oxidative stress that correlated with increased nitrotyrosine-positive islets in the pancreas (Figs. 1A-C), as previously reported (19). On the other hand, insulitis and nitrotyrosine staining were markedly reduced in NOD PI transgenic mice at 12 weeks of age (Figs. 1A-C). Enhanced nitrotyrosine staining is indicative of oxidative stress, and results from the increased production of superoxide and nitric oxide and the generation of peroxynitrite. Therefore we next examined whether the increased ROS promote PTP oxidation and inactivation in the pancreata of NOD mice. To this end, we used an antibody (PTPox) developed against the signature motif of the prototypic PTPN1/PTP1B oxidized to the irreversible sulfonic (-SO3H) state (9; 20). This antibody can detect the majority of classical PTPs when oxidized to the sulfonic state. Pancreata from 12 week-old NOD and NOD PI mice were homogenized in the presence of Nethylmaleimide to prevent post-lysis oxidation and to alkylate all reduced and active PTPs. This was followed by the reduction of reversibly oxidized PTPs and their subsequent hyperoxidation to the -SO3H state for detection with PTPox by immunoblot analysis (Fig. 1D). In pancreata from NOD mice we detected an increase in the oxidation status of several phosphatases; these included PTPox species with molecular masses of approximately 45/48 kDa, 50 kDa and 67 kDa. These bands co-migrate with the phosphatases PTPN2, PTPN1 and PTPN6 respectively. Importantly, there were no significant differences in the total expression of these PTPs in NOD and NOD PI mice
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(Fig. 1D). We monitored for the oxidation status of PTPN2 and PTPN6 proteins by immunoprecipitation (Fig. 1E, Supplementary Fig. 1A). We found PTPN2 and PTPN6 in PTPox immunoprecipitates from NOD mice (Fig. 1E) consistent with increased oxidation. We next determined if the oxidation of PTPs in immune-infiltrated pancreata was in fact mediated by the enhanced ROS levels. We found that treating NOD mice with the anti-oxidants N-acetyl cysteine (NAC) or mito-TEMPO (mTEMPO) in the drinking water attenuated PTP oxidation (Fig. 1F). Moreover, mTEMPO decreased immune infiltration (Fig. 1G).
STAT1 is a direct substrate of PTPN2 and this PTP has been implicated in β-cell function and survival (21; 22). Given the systemic PTP oxidation evident in NOD mice, we next asked whether the increased ROS promote PTP oxidation and inactivation to exacerbate STAT1 signaling. We therefore isolated pancreas from 12 week-old NOD and NOD PI mice and measured pSTAT1 levels by immunofluorescence. In keeping with a previous study (23), we observed increased STAT1 activation in immuneinfiltrated islets from NOD mice (Fig. 1H, Supplementary Fig. 1B).
Inactivation of PTPs increases IFN-γ signaling in islets
To study whether PTP inactivation enhances IFN-γ signaling and STAT1 activation in β-cells, we used the reversible PTP inhibitor sodium orthovanadate (Na3VO4). MIN6 and NIT-1 cells were treated with the PTP inhibitor and IFN-γ, and the effect of PTP inhibition on the kinetics and magnitude of IFN-γ-induced STAT1 phosphorylation was evaluated. STAT1 phosphorylation was highly induced after IFN-γ treatment in both controls and PTP inactivated cells (Figs. 2A-B). Although the phosphorylation of STAT1 occurred with different kinetics in the β-cell lines, it was markedly prolonged
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in cells in which PTPs were inhibited with Na3VO4 (Figs. 2A-B). Comparable results were observed in mouse islets treated with Na3VO4 or a second PTP inhibitor (PTPXVIII), and in human islets treated with Na3VO4 (Figs. 2C-D).
During insulitis, locally produced cytokines can both contribute to β-cell apoptosis (1) and stimulate the production of several chemokines through STAT1 activation in islets (24). PTP inactivation enhanced the expression of the STAT1-regulated chemokines CXCL9, CXCL10, and CXCL11 in mouse islets (Fig. 2E).
Enhanced IFN signaling induces β -cell death
We next evaluated whether PTP inhibition affects IFN-induced apoptosis in β-cells and primary islets. MIN6 cells were treated with a combination of the PTP inhibitor and IFN-γ (Fig. 3A). None of the treatments alone significantly affected cell viability, whereas PTP inhibition exacerbated apoptotic cell death in IFN-γ-treated cells (Fig. 3A). Importantly, we confirmed this result in human islets and β-cells and in mouse primary islets (Figs. 3B-D, Supplementary Figs. 2A-D). To determine the death pathways by which PTP inactivation exacerbates IFN-γ-induced cell death, we first analyzed the STAT1 signaling pathway, previously shown to be associated with cytokine-induced apoptosis of β-cells (1). Islets lacking STAT1 expression or overexpressing the suppressor of cytokine signaling (SOCS)-1 in β-cells were significantly protected against cell death induced by PTP inhibition and IFN-γ treatment (Fig. 3D).
We next examined Bcl-2 modulators of the intrinsic mitochondrial pathway of apoptosis. The focus was on the BH3-only proteins Bim and p53 upregulated modulator of apoptosis (PUMA) because these molecules have been implicated in the
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mechanism of cytokine-mediated β-cell death (2; 22). Islets derived from PUMA knockout mice were not protected from cell death (Fig. 3E). On the other hand, Bim inactivation significantly reduced cell death induced by PTP inactivation and IFN-γ treatment (Fig. 3E), suggesting a major role for this pro-apoptotic protein.
Type I IFN has been associated with the development of autoimmune diabetes in diabetic patients and in NOD mice (22). Thus, we examined the effect of PTP inactivation in mouse islets treated with IFN-α. Similar to IFN-γ, IFN-α induced cell death after PTP inactivation (Fig. 3F). The effect was specific since islets deficient in IFN-α receptors (IFNAR1-/-) were protected from cell death (Fig. 3F).
To directly assess the role of ROS in IFN-γ signaling, we used mouse pancreata deficient for the cytosolic and mitochondrial antioxidant enzyme glutathione peroxidase 1 (GPx1) that converts H2O2 to water. GPx1-deficiency results in elevated H2O2
levels and
oxidative
stress
and
increases pancreatic PTP oxidation
(Supplementary Fig. 3;(25)). In line with these data, isolated islets from GPx1 knockout mice had enhanced STAT1 activation after IFN-γ treatment (Fig. 3G). Moreover, GPx1 knockout islets were highly sensitive to IFN-γ, and cell death was prevented with antioxidant treatment (Fig. 3H). Taken together these results suggest that oxidative
stress causes the inactivation of PTPs, to enhance IFN signaling and the inflammatory response to promote cell death in pancreatic islets.
DISCUSSION
The present study demonstrates for the first time that PTPs are inactivated upon immune-infiltration to the pancreas, acting as an upstream event of the IFN signaling
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pathway in islets in T1D. This builds on our recently published work showing the in vivo inactivation of PTPs by oxidative stress in obesity and insulin resistance (9).
Pancreatic β-cells express very low levels of antioxidant enzymes and are extremely sensitive to oxidative stress induced by inflammation (12). Inhibition of NADPH oxidases such as NOX1 that produces superoxide protects β-cells from toxic effects of inflammatory cytokines (26). However, the mechanism(s) linking ROS to the promotion of inflammation and thereby β-cell death remain(s) unclear. Here, we demonstrated that insulitis results in PTP oxidation in the pancreas of pre-diabetic NOD mice. Furthermore, global inactivation of PTPs in the islets, results in increased pSTAT1 and downstream targets of IFNs and contributes to the β-cell’s own demise. The JAK/STAT pathway modulates immune-mediated β-cell dysfunction and death. In line with our results, deficiency of STAT1 in NOD mice prevents islet inflammation (3), and protects β-cells against immune-mediated destruction induced by multiple low doses of streptozotocin (4). Moreover, overexpression of SOCS-1 in β-cells inhibits IFN signaling and protects NOD mice from insulitis and diabetes (5). Interestingly, one of the PTPs oxidized in NOD pancreas is PTPN2, the inactivation of which has been shown in previous studies to enhance STAT1 phosphorylation and sensitize β-cells to apoptosis induced by IFNs (8; 21; 22). Consistent with our data, silencing of the pro-apoptotic molecule Bim prevents β-cell death induced by knockdown of PTPN2 and IFN treatment (22). In humans, genome-wide association studies have linked PTPN2 polymorphisms with T1D (27). Thus, our results establish an important novel mechanism of inactivation of T1D candidate genes by oxidative stress during insulitis. Indeed, overexpression of specific PTPs (e.g. PTPN2) might be a valuable strategy to protect β-cells against immune infiltration. However, if the
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phosphatase active site is inactivated by oxidative stress, as we demonstrate in the present study, combined therapies to overexpress PTPs and prevent ROS formation might be a better and more effective strategy to prevent β-cell destruction. Understanding which specific PTP to overexpress will be the subject of future work.
Although in this study we focused our attention on PTP inactivation in islets and βcells, it is probable that the oxidation and inactivation of PTPs in immune cells also contributes to the inflammatory process of T1D. For example, CD8+ T cells deficient for PTPN2 and cross-primed by β-cell self-antigens escape tolerance and acquire cytotoxic T cell activity, resulting in β-cell destruction in the RIP-mOVA model of autoimmune diabetes (28). Nevertheless, the role of global PTP oxidation/inactivation in immune cells during insulitis remains to be determined.
Dying β-cells may act as a “danger signal” in early T1D and, together with the local release of pro-inflammatory cytokines and chemokines, induce the amplification of the autoimmune reaction. We presently demonstrated that PTP inactivation plays a key role for β-cell death in the context of IFN signaling. Our research highlights the potential for oxidative stress and PTP oxidation to drastically alter cellular signaling in pathology (i.e. T1D), which has relevant implications for the development of effective treatments of the disease.
ACKNOWLEDGMENTS
We thank L Elkerbout, L Yachou-Wos, S Fynch, S Thorburn, C Selck for technical assistance, and Dr T Loudovaris and Ms L Mariana (Australian Islet Transplant Consortium, St Vincent’s Institute) for human islets. The authors declare no conflict of interest.
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FUNDING
This work was supported by a National Health and Medical Research Council of Australia (NHMRC) project grant (APP1071350) and fellowship (HET). ENG is supported by a Juvenile Diabetes Research Foundation (JDRF) fellowship. The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.
AUTHOR CONTRIBUTIONS
WS, SAL, HSQ, SMT researched data. TWHK, TT, JBdH and HET contributed to experimental design and discussion and reviewed and edited the manuscript. ENG researched data; contributed to discussion; designed experiments; and reviewed, edited, and wrote the manuscript. ENG is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
REFERENCES
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6. Ostman A, Frijhoff J, Sandin A, Bohmer FD. Regulation of protein tyrosine phosphatases by reversible oxidation. Journal of biochemistry 2011;150:345-356 7. Tiganis T, Bennett AM. Protein tyrosine phosphatase function: the substrate perspective. The Biochemical journal 2007;402:1-15 8. Gurzov EN, Stanley WJ, Brodnicki TC, Thomas HE. Protein tyrosine phosphatases: molecular switches in metabolism and diabetes. Trends in endocrinology and metabolism: TEM 2014; 9. Gurzov EN, Tran M, Fernandez-Rojo MA, Merry TL, Zhang X, Xu Y, Fukushima A, Waters MJ, Watt MJ, Andrikopoulos S, Neel BG, Tiganis T. Hepatic Oxidative Stress Promotes Insulin-STAT-5 Signaling and Obesity by Inactivating Protein Tyrosine Phosphatase N2. Cell metabolism 2014;20:85-102 10. Maxwell SR, Thomason H, Sandler D, Leguen C, Baxter MA, Thorpe GH, Jones AF, Barnett AH. Antioxidant status in patients with uncomplicated insulin-dependent and non-insulin-dependent diabetes mellitus. Eur J Clin Invest 1997;27:484-490 11. Rocic B, Vucic M, Knezevic-Cuca J, Radica A, Pavlic-Renar I, Profozic V, Metelko Z. Total plasma antioxidants in first-degree relatives of patients with insulindependent diabetes. Exp Clin Endocrinol Diabetes 1997;105:213-217 12. Lenzen S. Oxidative stress: the vulnerable beta-cell. Biochemical Society transactions 2008;36:343-347 13. Campbell PD, Weinberg A, Chee J, Mariana L, Ayala R, Hawthorne WJ, O'Connell PJ, Loudovaris T, Cowley MJ, Kay TW, Grey ST, Thomas HE. Expression of pro- and antiapoptotic molecules of the Bcl-2 family in human islets postisolation. Cell transplantation 2012;21:49-60 14. McKenzie MD, Dudek NL, Mariana L, Chong MM, Trapani JA, Kay TW, Thomas HE. Perforin and Fas induced by IFNgamma and TNFalpha mediate beta cell death by OT-I CTL. Int Immunol 2006;18:837-846 15. Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M, Cardozo AK, Eizirik DL. Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis. Cell death and differentiation 2009;16:1539-1550 16. Ichii H, Inverardi L, Pileggi A, Molano RD, Cabrera O, Caicedo A, Messinger S, Kuroda Y, Berggren PO, Ricordi C. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2005;5:1635-1645 17. McKenzie MD, Jamieson E, Jansen ES, Scott CL, Huang DC, Bouillet P, Allison J, Kay TW, Strasser A, Thomas HE. Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax. Diabetes 2010;59:644-652 18. French MB, Allison J, Cram DS, Thomas HE, Dempsey-Collier M, Silva A, Georgiou HM, Kay TW, Harrison LC, Lew AM. Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice. Diabetes 1997;46:34-39 19. Reddy S, Bradley J. Immunohistochemical demonstration of nitrotyrosine, a biomarker of oxidative stress, in islet cells of the NOD mouse. Annals of the New York Academy of Sciences 2004;1037:199-202 20. Persson C, Kappert K, Engstrom U, Ostman A, Sjoblom T. An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases. Methods 2005;35:37-43 21. Moore F, Colli ML, Cnop M, Esteve MI, Cardozo AK, Cunha DA, Bugliani M, Marchetti P, Eizirik DL. PTPN2, a candidate gene for type 1 diabetes, modulates
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interferon-gamma-induced pancreatic beta-cell apoptosis. Diabetes 2009;58:12831291 22. Santin I, Moore F, Colli ML, Gurzov EN, Marselli L, Marchetti P, Eizirik DL. PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic beta-cell apoptosis via regulation of the BH3-only protein Bim. Diabetes 2011;60:3279-3288 23. Suk K, Kim S, Kim YH, Kim KA, Chang I, Yagita H, Shong M, Lee MS. IFNgamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death. Journal of immunology 2001;166:4481-4489 24. Moore F, Naamane N, Colli ML, Bouckenooghe T, Ortis F, Gurzov EN, IgoilloEsteve M, Mathieu C, Bontempi G, Thykjaer T, Orntoft TF, Eizirik DL. STAT1 is a master regulator of pancreatic {beta}-cell apoptosis and islet inflammation. The Journal of biological chemistry 2011;286:929-941 25. Merry TL, Tran M, Stathopoulos M, Wiede F, Fam BC, Dodd GT, Clarke I, Watt MJ, Andrikopoulos S, Tiganis T. High-fat-fed obese glutathione peroxidase 1deficient mice exhibit defective insulin secretion but protection from hepatic steatosis and liver damage. Antioxidants & redox signaling 2014;20:2114-2129 26. Weaver JR, Holman TR, Imai Y, Jadhav A, Kenyon V, Maloney DJ, Nadler JL, Rai G, Simeonov A, Taylor-Fishwick DA. Integration of pro-inflammatory cytokines, 12-lipoxygenase and NOX-1 in pancreatic islet beta cell dysfunction. Molecular and cellular endocrinology 2012;358:88-95 27. Barrett JC, Clayton DG, Concannon P, Akolkar B, Cooper JD, Erlich HA, Julier C, Morahan G, Nerup J, Nierras C, Plagnol V, Pociot F, Schuilenburg H, Smyth DJ, Stevens H, Todd JA, Walker NM, Rich SS, Type 1 Diabetes Genetics C. Genomewide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nature genetics 2009;41:703-707 28. Wiede F, Ziegler A, Zehn D, Tiganis T. PTPN2 restrains CD8 T cell responses after antigen cross-presentation for the maintenance of peripheral tolerance in mice. Journal of autoimmunity 2014;53:105-114
FIGURE LEGENDS
Figure 1. Oxidative stress and pancreatic PTP oxidation in pre-diabetic NOD mice. A: Insulitis development was scored on pancreata collected from female NOD PI and NOD mice at 12 weeks of age. Serial sections (3-µm thick) were prepared at three levels (200 µm apart) and scored in a blinded manner using the following scale: 0 = no infiltrate, 1 = peri-islet infiltrate, 2 = extensive (>50%) peri-islet infiltrate, 3 = intraislet infiltrate, and 4 = extensive intraislet infiltrate (>80%) or total β-cell loss. Greater than 50 islets per pancreas were scored. The percentage of islets per pancreas with each score was calculated. Scores were added to give an overall score for each
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pancreas, and data are shown as the mean. B: Paraffin sections (3 µm) of pancreas from 12 week-old NOD PI and NOD mice stained with hematoxylin and eosin (H&E). C: Paraffin sections were immunolabeled with the oxidative stress marker nitrotyrosine and counterstained with hematoxylin. D: Immunoblot analysis with PTPox antibody, to determine relative PTP oxidation (PTP-SO3H) or for the indicated PTPs in 12-week old NOD PI versus NOD pancreas samples. E: Immunoprecipitation (IP) of PTPox proteins from NOD pancreas samples and immunoblotting for PTPN6
(67 kDa) or PTPN2 (45/48 kDa). IgG heavy chain (HC) band (50 kDa) is shown. F: Six week-old female NOD mice were treated with NAC (Sigma, St. Louis, MO, 1mg/ml) for 8 weeks or mTEMPO (Enzo Life Sciences, Farmingdale, NY, 1mM) for 14 weeks in drinking water and pancreata extracted for an assessment of PTP oxidation by SDS-PAGE. G: Insulitis scores on pancreata collected from female NOD mice either
control treated, or treated with mTEMPO for 20 weeks (n=8 per group). H: Cryosections of pancreas from 12 week-old NOD PI or NOD mice stained with antibodies recognizing pSTAT1, insulin and DAPI (Bar, 50 µm). Quantification of pSTAT1 staining after correction for nuclear DAPI in islets is shown. A total of 25 islets from 4 NOD PI mice, 7 islets without infiltration and 124 islets with infiltration from 6 NOD mice were scored. *P < 0.05, ***P < 0.001. Confocal image of overlapped staining (green: pSTAT1, red: insulin, blue: DAPI) is shown to demonstrate pSTAT1/insulin co-staining (white arrows; Bar, 10 µm).
Figure 2. IFN-γ-induced STAT1 signaling in β-cells and pancreatic islets. A-B: Time course of STAT1 protein activation after IFN-γ (100 U/ml, BioLegend, CA) and PTP inactivation with Na3VO4 (100 µM, Sigma) in MIN6 (A) or NIT-1 (B) cells. Cell lysates were subjected to Western blotting with antibodies detecting pSTAT1, STAT1 or β-actin as loading control. The intensity values for the proteins were
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corrected by the values of the housekeeping protein β-actin and are shown as arbitrary units (A.U.). Results are the means ± SEM of 3-5 independent experiments. *P < 0.05. C: Western blot demonstrating activation of STAT1 in mouse islets treated for 8h with IFN-γ (100 U/ml), Na3VO4 (100 µM), the PTP inhibitor XVIII (PTPXVIII, 1 µM, Millipore) or combination as indicated. The result is representative of two independent experiments. D: Activation of STAT1 in human islets isolated from two organ donors following treatment with IFN-γ (100 U/ml) and/or Na3VO4 (100 µM) for 24h. E: Mouse islets were treated with IFN-γ, Na3VO4, or combination for 8h. qRT-PCR for STAT1-dependent chemokines and β-actin expression was then performed. Individual chemokine values have been divided by the housekeeping gene β-actin and presented as fold induction related to the Na3VO4 treated samples (considered as 1). Results are the means ± SEM of 4 independent experiments. *P < 0.05, **P < 0.01.
Figure 3. Inactivation of PTPs potentiates cell death induced by IFNs. A: Cell death of MIN6 cells was evaluated by Hoechst-33342 (blue)/propidium iodide (red) 24 h after IFN-γ (100 U/ml) and PTP inactivation with Na3VO4 (100 µM) as indicated. Arrows indicate PI positive cells. Data shown are means ± SEM of 4 independent experiments. **P < 0.01. B: Human islets were cultured for 48h with IFN-γ (100 U/ml), Na3VO4 (100 µM), or combination, and viability was measured by 7aminoactinomycin D (7-AAD) staining. Data shown are means ± SEM of 3 independent experiments. *P < 0.05. C: Human β-cells from two organ donors treated with IFN-γ (100 U/ml), Na3VO4 (100 µM), or combination for 48h. Human islets were dispersed into single cells and stained with 7-AAD, NG and TMRE. After gating out 7-AAD+ cells, NG positive cells (β-cells) were then analyzed for their percentages of TMRE+ cells (viability). D: DNA fragmentation was measured by
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flow cytometry in islets from wild-type, STAT1-/-, or RIP-SOCS1 mice cultured in medium containing IFN-γ, Na3VO4, or combination for 24h. Data shown are means ± SEM of 4 independent experiments. *P < 0.05, **P < 0.01. E: DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Puma-/- or Bim-/- islets for 24h with IFN-γ, Na3VO4, or combination. Data shown are means ± SEM of 4 independent experiments. *P < 0.05. F: DNA fragmentation was measured by flow cytometry in islets from wild-type, or IFNAR1-/- mice cultured in medium containing IFN-α (PBL Interferon Source, NJ), Na3VO4, or combination for 24h. Data shown are means ± SEM of 3 independent experiments. **P < 0.01. G: Mouse islets from GPx1+/+ and GPx1–/– mice were isolated, treated with IFN-γ for 24h and processed for immunoblot analysis. H: DNA fragmentation was measured by flow cytometry after incubation of GPx1+/+ and GPx1–/– islets for 24h with IFN-γ (100 U/ml), NAC (1 mM), or combination. Data shown are means ± SEM of 3 independent experiments. *P < 0.05.
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Diabetes
B
100%
Grade 4 Grade 3
NOD PI
NOD
Grade 2
60%
Grade 1 40%
Grade 0
C
20% 0%
D
NOD
Nitrotyrosine
Insulitis
80%
NOD PI H&E
A
NOD PI 1 2 3
E
NOD 1 2 3 4
IP: control 1
5
2
3 4
F
IP: PTPox 1
2
3
4
NOD control
PTPN6 98 kDa
IgG (HC)
1 2 3
NOD NOD control mTEMPO
NOD NAC 1 2
3
1
2
1
2
65 kDa 50 kDa
65kDa
65 kDa
PTPox IgG (HC) 36 kDa
50 kDa
50 kDa
36 kDa
36 kDa
PTPN2 PTPox
G
PTPN6
65 kDa
PTPN1
50 kDa
PTPN2
50 kDa
Insulitis
actin
100%
Grade 4
80%
Grade 3
60%
Grade 2 Grade 1
40%
Grade 0
20%
actin Pancreas homogenates from individual mice
0%
actin Pancreas homogenates from individual mice
NOD PI
50μm
NOD
0.300
pSTAT1/DAPI
pSTAT1
H
*** ***
0.200
* 0.100
Insulin
0.000
DAPI
NOD (confocal)
10μm
Figure 1
Diabetes
MIN6 Cells
control
Na3VO4
Incubation time with IFN-γ: 0h 2h 4h 8h 24h
0.8
0h 2h 4h 8h 24h
pSTAT1/actin (A.U.)
A
pSTAT1 STAT1 actin
B
Page 20 of 27
control Na3VO4
0.6
*
0.4
* 0.2 0 0h
NIT-1 Cells
pSTAT1
STAT1
1
8h
*
0.6 0.4 0.2 0 0h
D
Mouse islets
2h
4h
8h
Na3VO4
IFN-γ +
IFN-γ
PTPXVIII IFN-γ +
Na3Vo4
donor 2
IFN-γ
Na3VO4
IFN-γ +
IFN-γ
pSTAT1
pSTAT1
STAT1
STAT1
STAT1
STAT1
actin
actin
actin
actin
*
1000
ddct (fold induction)
ddct (fold induction)
CXCL10 100
800 600 400 200 IFN-γ
Na3VO4
*
80 60 40 20 0
0 IFN-γ +
IFN-γ
Na3VO4
1 0 IFN-γ
Na3VO4
IFN-γ + Na3VO4
ddct (fold induction)
ddct (fold induction)
ddct (fold induction)
**
5
IFN-γ
Na3VO4
IFN-γ +
4 3 2 1 0 IFN-γ
Na3VO4
CXCL2
5
5
2
10
Na3VO4
CXCL1
3
15
Na3VO4
CCL20
4
*
20
0
IFN-γ +
Na3VO4
5
CXCL11 25 ddct (fold induction)
CXCL9
1200
IFN-γ + Na3Vo4
pSTAT1
Mouse islets
Na3VO4
Na3Vo4
PTPXVIII
pSTAT1
E
24h
Human islets donor 1
IFN-γ
24h
0.8
actin
C
4h
control Na3VO4
1.2
pSTAT1/actin (A.U.)
control Na3VO4 Incubation time with IFN-γ: 0h 2h 4h 8h 24h 0h 2h 4h 8h 24h
2h
IFN-γ + Na3VO4
4 3 2 1 0
IFN-γ
Na3VO4
IFN-γ + Na3VO4
Figure 2
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A
Diabetes
Control
Na3VO4
B
MIN-6 cells
Human islets
25
100
**
IFN-γ + Na3VO4
*
80 Viability (%)
IFN-γ
Cell Death (%)
20
15
10
60
40
20
5
0
0 Control
IFN-γ Na3VO4 IFN-γ +
IFN-γ
Na3VO4 IFN-γ +
Na3VO4
Donor 1
40
80
30
IFN-γ + Na3VO4
Na3VO4
0
IFN-γ
IFN-γ + Na3VO4
Na3VO4
IFN-γ
0
DNA Fragmentation (%)
45
15
20
F
DNA Fragmentation (%)
60 Viability (%)
Viability (%)
80 60
100
Donor 2
IFN-α (100 U/ml) Na3VO4 (100 μM) IFN-α (100 U/ml) + Na3VO4 (100 μM)
IFN-γ (100 U/ml) Na3VO4 (100 μM) IFN-γ (100 U/ml) + Na3VO4 (100 μM)
**
80
* 60 40 20
80
0
IFN-γ (100 U/ml) Na3VO4 (100 μM) IFN-γ (100 U/ml) + Na3VO4 (100 μM)
60
*
40
20
0 Wild-type
STAT1 -/-
RIPSOCS1
G
Wild-type
H 30
IFN-γ (100 U/ml) GPx1 +/+
60
**
1 pSTAT1
40
E DNA Fragmentation (%)
D
Human β-cells
STAT1 20
2
GPx1 -/1
2
DNA Fragmentation (%)
C
Na3VO4
PUMA -/-
Bim -/-
Control IFN-γ (100 U/ml) IFN-γ (100 U/ml) + NAC (1 mM)
25
*
20 15 10 5
actin 0
0 Wild-type
IFNAR1-/-
GPx1 +/+
GPx1 -/-
Figure 3
Diabetes
Supplemental Data Inactivation of protein tyrosine phosphatases enhances interferon signaling in pancreatic islets. William J. Stanley, Sara A. Litwak, Hong Sheng Quah, Sih Min Tan, Thomas W. H. Kay, Tony Tiganis, Judy B. de Haan, Helen E. Thomas, Esteban N. Gurzov
Supplementary Figure 1. A. Immunoprecipitation (IP) of PTPox proteins from NOD PI and NOD pancreas samples and immunoblotting for PTPN2. IgG heavy chain (HC, 50 kDa) and PTPN2 bands are shown. B. Confocal microscopy for pSTAT1 (green), insulin (red) and DNA (blue) in pancreatic islets from 12 week-old NOD PI and NOD mice. White arrows indicate pSTAT1 activation in insulin positive cells.
1
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Diabetes
Supplementary Figure 2. A. Human islets were dispersed into single cell suspensions and stained with 7-AAD. Representative FACS profiles showing the percentage of 7-AAD+ (dead cells) in human islets treated with IFN-γ, Na3VO4, or combination for 48h. Data is representative of 3 independent experiments and quantification presented in Fig. 3B. B. Representative FACS profiles of human β-cells treated with IFN-γ, Na3VO4, or combination for 48h, dispersed into single cells and stained with 7-AAD, NG and TMRE. After gating out 7AAD+ cells, NG positive cells (β-cells) were then analyzed for their percentages of TMRE+ (alive) and TMRE- (apoptotic) cells. Data shown is representative of two independent experiments presented in Fig. 3C. C-D. Representative FACS profiles of mouse islets treated with IFN-γ, Na3VO4, or combination for 24h. The percentage of islet cells with fragmented nuclei is indicated. Data shown is representative of 4 independent experiments and quantifications presented in Figs. 3D-E. 2
Diabetes
Supplementary Figure 3. Immunoblot analysis with PTPox antibody to determine relative PTP oxidation (PTP-SO3H) in pancreas samples from16 week-old GPx1+/+ and GPx1–/– mice.
3
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Page 25 of 27
Diabetes
Mice NOD
C57BL/6
NOD PI STAT1-/RIP-SOCS-1 Bim-/-
Puma-/-
NOD.IFNAR1−/− GPx1-/-
Reference Walter and Eliza Hall Institute of Medical Research (Melbourne, VIC, Australia) Walter and Eliza Hall Institute of Medical Research (Melbourne, VIC, Australia) (1) (generated on a NOD background) (2) (3) (generated on a NOD background) (4; 5) (generated using 129SV-derived ES cells and backcrossed for >10 generations onto the C57BL/6 background) (5; 6) (generated using C57BL/6-derived ES cells) (7) (8; 9)
Supplementary Table 1. List of mice used for in vivo studies and islet isolation.
Probe
CXCL9 (mouse) CXCL10 (mouse) CXCL11 (mouse) CCL20 (mouse) CXCL1 (mouse) CXCL2 (mouse) β-actin (mouse)
Catalogue number (Applied Biosystems, Foster City, CA, USA) Mm00434946_m1
Mm00445235_m1
Mm00444662_m1
Mm01268754_m1
Mm04207460_m1
Mm00436450_m1
Mm00607939_s1
Supplementary Table 2. List of probes used for qPCR. Real-time PCR was performed using the Rotor-Gene RG-3000 machine (Corbett Research; Qiagen, Hilden, Germany) and the TaqMan PCR Master Mix (AmpliTaq Gold with GeneAmp kit; Applied Biosystems) in 20 µl reaction volumes.
4
Diabetes
Antibody
PTPox STAT1
pSTAT1
PTPN6
PTPN1
PTPN2
β-Actin
Company R&D Systems, Minneapolis, MN BD Biosciences, CA
Reference
Dilution
MAB2844 #610115
1/1000 1/500
BD Biosciences, CA Cell Signaling, Danvers, MA
#612132
1/1000
#3759
1/1000
BD Biosciences, CA R&D Systems, Minneapolis, MN Santa Cruz Biotechnology, CA
610139
1/500
MAB1930
1/1000
sc-7210
1/5000
12-348
1/5000
P0260
1/10000
HRP-conjugated anti-rabbit IgG Millipore, Temecula, CA
HRP-conjugated anti-mouse IgG
Page 26 of 27
DAKO, Denmark
Supplementary Table 3. List of antibodies used for Western blot analysis.
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Diabetes
EXTENDED REFERENCES. 1. French MB, Allison J, Cram DS, Thomas HE, Dempsey-Collier M, Silva A, Georgiou HM, Kay TW, Harrison LC, Lew AM. Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice. Diabetes 1997;46:34-39 2. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 1996;84:431-442 3. Chong MM, Chen Y, Darwiche R, Dudek NL, Irawaty W, Santamaria P, Allison J, Kay TW, Thomas HE. Suppressor of cytokine signaling-1 overexpression protects pancreatic beta cells from CD8+ T cell-mediated autoimmune destruction. Journal of immunology 2004;172:5714-5721 4. Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM, Strasser A. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 1999;286:1735-1738 5. McKenzie MD, Jamieson E, Jansen ES, Scott CL, Huang DC, Bouillet P, Allison J, Kay TW, Strasser A, Thomas HE. Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax. Diabetes 2010;59:644652 6. Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM, Strasser A. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 2003;302:1036-1038 7. Quah HS, Miranda-Hernandez S, Khoo A, Harding A, Fynch S, Elkerbout L, Brodnicki TC, Baxter AG, Kay TW, Thomas HE, Graham KL. Deficiency in type I interferon signaling prevents the early interferon-induced gene signature in pancreatic islets but not type 1 diabetes in NOD mice. Diabetes 2014;63:1032-1040 8. de Haan JB, Bladier C, Griffiths P, Kelner M, O'Shea RD, Cheung NS, Bronson RT, Silvestro MJ, Wild S, Zheng SS, Beart PM, Hertzog PJ, Kola I. Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide. The Journal of biological chemistry 1998;273:22528-22536 9. Tan SM, Stefanovic N, Tan G, Wilkinson-Berka JL, de Haan JB. Lack of the antioxidant glutathione peroxidase-1 (GPx1) exacerbates retinopathy of prematurity in mice. Investigative ophthalmology & visual science 2013;54:555-562
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