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Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice
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ARTICLE IN PRESS Available online at
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Short report
Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice M.S. Wong a , A. Tso a , M. Ali a , W.J. Hawthorne b , N. Manolios a,∗ a b
Rheumatology Department, The University of Sydney/Westmead Hospital, Westmead NSW 2145, Australia Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead NSW 2145, Australia Received 19 December 2013; received in revised form 25 January 2014; accepted 31 January 2014
Abstract The aim of this study was to investigate the effectiveness of immunomodulatory peptides in preventing the spontaneous onset of Type 1 diabetes in NOD mice. Two such peptides, CP and C1, were injected intraperitoneally in NOD mice three times a week starting at two different time points, nine weeks and 11 weeks of age, and blood sugar levels monitored for the development of diabetes. CP was shown to be effective in delaying the onset of diabetes compared to control (P = 0.006). The timing of peptide administration was crucial since delay in treatment did not prevent the onset of diabetes (nine weeks versus 11 weeks of age). C1 was effective in delaying the onset of Type 1 diabetes with borderline significance when given at week 11 (P = 0.05). These findings confirm the efficacy of these peptides in the prevention and possible treatment for Type 1 diabetes and thereby create new opportunities for genetic manipulation. © 2014 Elsevier Masson SAS. All rights reserved. Keywords: Immunosuppression; Non-obese diabetic (NOD) mouse; Peptide; Type 1 diabetes (T1D); Core peptide
1. Introduction Type 1 diabetes (T1D) is an autoimmune T-cell mediated disease leading to elevated blood glucose levels (BGLs). T1D is a common disease affecting 87,100 individuals in Australia, who require daily insulin injections [1]. These individuals must also monitor their BGLs, diet and exercise regularly to ensure control of their diabetes. Unfortunately, many of these patients will subsequently develop secondary complications from their disease and place an enormous personal and financial burden on the community and government. In Australia, the total annual cost (healthcare costs, the cost of carers and Commonwealth government subsidies) is $570 million, with $4669 being the average annual cost per person [2]. For individuals that have not developed secondary complications, the average annual cost is $3468, however if secondary micro- and macro-vascular complications develop, the average annual cost rises to $16,698 [2]. Several potential treatment options for patients with endstage diabetic renal failure include simultaneous pancreas and kidney transplantation; and more recently, to a subgroup of very
∗
Corresponding author. Tel.: +61 2 98458099; fax: +61 2 98458317. E-mail address: [email protected] (N. Manolios).
brittle diabetics transplantation of islets [3,4]. The problem with both of these is rejection of the graft or islets, and the secondary complications arising from the use of immunosuppressive therapy, such as prednisone, tacrolimus and cyclosporin. Newer approaches and agents such as interleukin (IL)-18 and IL-7Ra inhibitors, have being used and examined with limited success [5,6]. Core peptide (CP; GLRILLLKV) is a novel immunosuppressive peptide that has the potential to treat T1D by inhibiting the activation of T-cells, and thus the T-cell mediated destruction of islets. CP consists of two basic positively charged and hydrophilic amino acids (arginine and lysine) separated by four neutral and hydrophobic amino acids [7]. The functional significance of this peptide was published in 1997 when it was shown that CP inhibited T-cell activation in vivo and in vitro [8]. In animal studies, CP given subcutaneously significantly reduced the induction of T-cell mediated inflammation in adjuvant induced arthritis (AIA), cyclophosphamide-induced diabetes, experimental allergic encephalomyelitis (EAE) and delayed type contact hypersensitivity [8,9]. C1 ( ) is a cyclic peptide similar in sequence to CP which has alternating D-, L- amino acids, increasing the peptide’s stability and resistance to proteolytic
1262-3636/$ – see front matter © 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.diabet.2014.01.007
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
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degradation [10–12]. C1 consists of two basic positively charged and hydrophilic amino acids (arginine and lysine) separated by five neutral and hydrophobic amino acids. C1 has the same ability to inhibit T-cell activation and IL-2 production as CP and in comparative studies, C1 was shown to be more effective than CP in its ability to inhibit T-cells (P < 0.012) [12]. In animal models of asthma and arthritis, C1 has been shown to be effective in suppressing the immune response [12]. This study examined the effects of CP and C1 on the onset of diabetes in non-obese diabetic (NOD) mice. This model exploits the fact that approximately 60–80% of NOD mice spontaneously develop T1D by a similar mechanism seen in human T1D [13,14] and aims to address if CP and C1 can inhibit the onset and severity of diabetes. 2. Methods 2.1. Animals All experimental protocols and methods were approved by the Western Sydney Local Health District (WSLHD) Animal Ethics Committee (AEC) and all experiments were performed in accordance with its guidelines. Female non-obese diabetic (NOD) mice were obtained at 8 weeks of age from the Animal Resources Centre (Murdoch, WA, Australia) and housed in the WSLHD Animal facility located within Westmead Hospital. Food and water was given ad libitum. The mice were randomly placed into a control group (n = 22) and treatment groups. In one group CP was given from nine weeks of age (n = 19); and in the second group CP was given from 11 weeks of age (n = 19). A third group was given C1 from 11 weeks (n = 14). Mice were deemed diabetic when the BGLs ≥ 15 mM on two consecutive readings.
can describe a situation where the vital event has not occurred; in this case, a mouse that has not yet developed diabetes. 2.5. Insulitis scoring Histological analysis was performed on pancreatic tissue collected from control and CPW9 mice at 22 weeks. A proportion of pancreatic tissue in the CPW9 group was collected three weeks after treatment ceased (at 25 weeks) and insulitis was scored separately from tissue collected at 22 weeks. The severity of insulitis was scored for at least 10 islets in each group according to Jimeno et al. [15]. When there was no lymphocytic infiltration, the sample was assigned “0, no-insulitis”. When lymphocytic infiltrations were seen around the islet and up to 30% of the islet, the sample was assigned as “1, peri-insulitis”. “2, moderate insulitis” was assigned to samples where there were lymphocytic infiltrations between 30% and 50% of the islet. Samples with lymphocytic infiltrations of more than 50% of the islet were assigned as “3, severe insulitis”. Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney tests were used to test for differences in insulitis scores and for pair-wise comparisons between groups, respectively. 3. Results As expected, 64% of the mice in the control group developed diabetes and only 36% of mice remained diabetes-free during a 13-week period of observation (week 9–22). The earliest onset
2.2. Treatment Peptides were dissolved in milliQ water and were sonicated for 30 min. The solutions were filter sterilised and stored at 4 ◦ C. CP was administered intraperitoneally (i.p.) at a concentration of 0.4 mg three times a week from either nine weeks or 11 weeks of age prior to the onset of diabetes. C1 was administered i.p. at a concentration of 0.1 mg three times a week from 11 weeks of age and the control group received no treatment. All treatments ceased at 22 weeks. 2.3. Monitoring BGLs Mice in all groups were fasted for at least 2 h prior to the measurement of their BGLs three times a week. 2.4. Kaplan-Meier survival plot A Kaplan-Meier survival plot was used to assess the percentage of NOD mice that developed diabetes over time. This statistical method is a common technique, performed to analyse survival-time data, when there is “censored” data. Censored data
Fig. 1. Kaplan-Meier survival plot comparing the number of female NOD mice that developed diabetes, with or without immunosuppressive peptide treatment. The control group (n = 22) is represented by the black solid line (A). Mice (n = 19) treated with 0.4 mg CP from 9 weeks of age is represented by the red solid line (B) and the mice (n = 19) treated with 0.4 mg CP from 11 weeks of age by the red dotted line (C). The mice (n = 14) treated with 0.1 mg C1 from 11 weeks of age is represented by the blue dotted line (D). By the end of the study, 14/22 (64%) mice in the control group had developed T1D. By contrast, only 4/19 (21%) mice developed diabetes in the group treated with CP from 9 weeks of age (B). In the group treated with CP from 11 weeks, 7/19 (37%) mice developed diabetes. Borderline significance (P = 0.05) was achieved when comparing the control group to the group treated with C1 from 11 weeks where 4/14 (29%) mice developed diabetes.
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
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of diabetes in the control group was at 11.4 weeks of age. By contrast in the CP treated group commencing at nine weeks of age (CPW9 ) 79% of the mice remained diabetes-free and only 21% developed diabetes (P = 0.006). In this treatment group, the onset of diabetes was delayed by four weeks compared to the control group. In the CP treated group commencing at 11 weeks
3
of age (CPW11 ) 63% of the mice remained diabetes-free and 37% of the mice developed diabetes, which was not significantly different from the control group. The earliest onset of diabetes noted in this treatment group was 11.4 weeks. In mice given C1 from 11 weeks of age (C1W11 ), 71% of mice remained diabetes-free while 29% of the mice developed
Fig. 2. Blood glucose levels of mice with or without immunosuppressive peptide treatment: a: untreated mice (n = 22); b: mice given CP from nine weeks of age (CPW9 , n = 19); c: CP given from 11 weeks of age (CPW11 , n = 19); d: mice given C1 from 11 weeks of age (C1W11 , n = 14).
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
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diabetes (P = 0.05) during an 11-week period of observation. The earliest onset of diabetes in the C1 treatment group was delayed by one week to 12.4 weeks. These results suggest that prophylactically administered CP was able to prevent the onset of T1D in NOD mice for more than 90 days. Any delay in treatment was not effective.
The Kaplan-Meier survival plot (Fig. 1) shows the number of mice that developed diabetes after three months observation and the BGL curves for each group are shown in Fig. 2. Histology of pancreatic tissue obtained from mice at the end of the study showed that CP ameliorated the T-cell mediated destruction of islets (Fig. 3). The stacked bar curve (Fig. 4) shows the
Fig. 3. Immunohistochemical staining of pancreatic islets: a: haematoxylin and eosin stain of pancreatic tissue depicting an islet; b: insulin stain within an islet; c: haematoxylin and eosin stain of pancreatic tissue from a mouse treated with CP that did not develop diabetes, depicting an islet (lower arrow) surrounded by lymphocytes (upper arrow); d: haematoxylin and eosin stain of pancreatic tissue from a mouse treated with CP that did not develop diabetes. Despite the lymphocytic infiltration the islets are still functional as depicted by the insulin stain.
Fig. 4. Stacked bar chart comparing the distribution of insulitis scores between control and CPW9 groups. The severity of insulitis was reduced (P = 0.010) in islets from the CPW9 group (n = 11) compared to islets in the control group (n = 41). No difference in insulitis (P = 0.076) was seen in islets from the CPW9 group regardless of whether the pancreas was collected immediately or three weeks after end of treatment (n = 80).
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
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distribution of insulitis scores between controls and the CPW9 pancreatic tissue collected three weeks apart. There was a statistical difference in insulitis scores between the three groups (P = 0.024). In particular, the insulitis scores in the control group were significantly higher than those in the CPW9 group (P = 0.01), indicating that treatment with CP reduced the severity of lymphocytic infiltration. Although it appeared that insulitis increased when CP was withheld for the final three weeks, there was no statistical difference between insulitis scores in the CPW9 (three weeks after end of Rx) group and the CPW9 group (P = 0.076). 4. Discussion The ability to prevent the onset of diabetes and maintain euglycaemia would have dramatic and significant effects in any T1D patient if achieved. The Diabetes Control and Complications Trial and Follow-up Study (DCCT) found that maintaining normal blood glucose levels reduces the risk of secondary complications [16]. There was a 76% decreased risk of eye disease, 50% reduced risk of kidney disease and a 60% reduced risk of nerve disease. A continuation of the DCCT, the Epidemiology of Diabetes Interventions and Complications study (EDIC) found that there was a 42% decreased risk in cardiovascular disease occurrence and a 57% reduced risk of nonfatal heart attack, stroke or death from cardiovascular causes [17]. Therefore, there is a significant advantage that even delaying the onset of T1D by a few years can dramatically reduce the incidence of secondary complications. In NOD mice, by the time mice are symptomatic at 12–14 weeks, 60–90% of the islet cell mass had already been destroyed [18]. By pre-treating NOD mice with CP from nine weeks of age, the onset of T1D could be delayed. As shown histologically, CP was able to influence the T-cell response with less cellular infiltrate and inflammation. CP given at 11 weeks of age did not delay the onset of disease. By contrast, C1 may be a more powerful immunosuppressive agent than CP in preventing the progression of T1D because when given at week 11 CP had no effect, however treatment with C1 was borderline significant compared to the control group (P = 0.05). CP acts on T-cells at the transmembrane level to inhibit signal transduction following antigen recognition [7]. Published studies have indicated that CP interferes with the interactions between TCR-␣, CD3␦ and dimers [9,19]. Prevention of a completely assembled TCR-CD3- complex inhibits signal transduction and activation of T-cells. By giving CP i.p. to NOD mice at an early age, it was hypothesised that CP will inhibit antigen activation of islet-targeting T-cells. The mechanism of action for C1 has not been clearly defined but presumed to be similar to CP. A limitation of the results could be the low animal numbers (14 mice in the C1 treated group) and increasing this number may possibly lead to a more significant difference compared to controls. Furthermore, treatment of mice with C1 from the earlier time point, nine weeks of age as in the CPW9 group, could influence the results. Administering treatment to coincide with the beginning of insulitis, which begins even earlier than nine weeks
5
of age, at three to four weeks of age in NOD mice [14], could dramatically improve the results and will be further investigated. Although there was a statistical significance noted between the BGLs of CP-treated and non-treated mice, further studies and longer periods of observation are needed. Subsequent studies could also include the analysis of the T-cell phenotype in the spleen and pancreatic lymph nodes of protected mice, measuring the changes in cytokine production by T-cells in the presence of CP and C1 as well as trialling C1 as an oral agent. Cyclosporin is also a cyclic peptide that is an immunosuppressant that has been used in the treatment of T1D [20,21]. Cyclosporin inhibits T-cell proliferation and the destruction of islets by blocking calcineurin and reducing IL-2 production [22]. Treatment with cyclosporin after clinical presentation of T1D has been shown to conserve insulin production, however the protective effects were not permanent [23,24]. Furthermore, the cyclosporin dose required to induce diabetes remission leads to nephrotoxicity and other adverse effects [21,25]. Development of new immunosuppressives, such as C1 and CP, could provide further drug choices in the treatment of T1D. Furthermore, the linear sequence of CP can be converted into cDNA, unlike cyclosporin, and may be used to induce tolerance when transduced into dendritic cells or provide a novel means of immunosuppression using gene therapy to transduce islets and prevent islet cell destruction. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments We express our sincere appreciation to our funding bodies – The Rebecca L. Cooper Medical Research Foundation, The Wenkart Foundation and Research Trust Fund of Rheumatology Dept., Westmead. We would also like to thank Ms Virginia James for assistance with the histology sections and Ms Karen Byth for statistical analysis. Appendix A. Supplementary data Supplementary data (French abstract) associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.diabet.2014.01.007. References [1] Australian Institute of Health and Welfare (AIHW). Diabetes prevalence in Australia: detailed estimates for 2007-08. Canberra: AIHW: 2011 Contract No.: Cat. no. CVD 56. [2] Colagiuri R. DiabCo$t type 1: assessing the burden of Type 1 diabetes in Australia. Canberra: A. C. T.: Diabetes Australia; 2009. p. 28. [3] Maffi P, Scavini M, Socci C, Piemonti L, Caldara R, Gremizzi C, et al. Risks and benefits of transplantation in the cure of Type 1 diabetes: whole pancreas versus islet transplantation. A single center study. Rev Diabet Stud 2011;8:44–50.
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
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[4] Light JA, Sasaki TM, Currier CB, Barhyte DY. Successful long-term kidney-pancreas transplants regardless of C-peptide status or race. Transplantation 2001;71:152–4. [5] Zaccone P, Phillips J, Conget I, Cooke A, Nicoletti F. IL-18 binding protein fusion construct delays the development of diabetes in adoptive transfer and cyclophosphamide-induced diabetes in NOD mouse. Clin Immunol 2005;115:74–9. [6] Lee LF, Logronio K, Tu GH, Zhai W, Ni I, Mei L, et al. Anti-IL-7 receptor-alpha reverses established Type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function. Proc Natl Acad Sci U S A 2012;109:12674–9. [7] Manolios N, Ali M, Amon M, Bender V. Therapeutic Applications of transmembrane T and natural killer cell receptor peptides. In: Sigalov A, editor. Multichain immune recognition receptor signalling: from spatiotemporal organization to human disease: Landes Bioscience. 2008. p. 208–19. [8] Manolios N, Collier S, Taylor J, Pollard J, Harrison LC, Bender V. T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease. Nat Med 1997;3:84–8. [9] Amon MA, Ali M, Bender V, Chan YN, Toth I, Manolios N. Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function. Biochim Biophys Acta 2006;1763:879–88. [10] Carmona G, Rodriguez A, Juarez D, Corzo G, Villegas E. Improved protease stability of the antimicrobial peptide pin2 substituted with d-amino acids. Protein J 2013;32:456–66. [11] Molhoek EM, van Dijk A, Veldhuizen EJ, Haagsman HP, Bikker FJ. Improved proteolytic stability of chicken cathelicidin-2 derived peptides by D-amino acid substitutions and cyclization. Peptides 2011;32:875–80. [12] Ali M, Amon M, Bender V, Bolte A, Separovic F, Benson H, et al. Cyclization enhances function of linear anti-arthritic peptides. Clin Immunol 2014;150:121–33. [13] Animal Resources Centre. Animal Resources Centre Animals and Services. [11/10/2013]; Available from: http://www.arc.wa.gov.au/service. php?category=Inbred+Mice
[14] Anderson MS, Bluestone JA. The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 2005;23:447–85. [15] Jimeno R, Gomariz RP, Gutierrez-Canas I, Martinez C, Juarranz Y, Leceta J. New insights into the role of VIP on the ratio of T-cell subsets during the development of autoimmune diabetes. Immunol Cell Biol 2010;88: 734–45. [16] Lasker RD. The diabetes control and complications trial. Implications for policy and practice. N Engl J Med 1993;329:1035–6. [17] Nathan DM, Cleary PA, Backlund JY, Genuth SM, Lachin JM, Orchard TJ, et al. Intensive diabetes treatment and cardiovascular disease in patients with Type 1 diabetes. N Engl J Med 2005;353:2643–53. [18] van Belle TL, Coppieters KT, von Herrath MG. Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev 2011;91:79–118. [19] Sigalov A. Multi-chain immune recognition receptors: spatial organization and signal transduction. Semin Immunol 2005;17:51–64. [20] Assan R, Feutren G, Debray-Sachs M, Quiniou-Debrie MC, Laborie C, Thomas G, et al. Metabolic and immunological effects of cyclosporin in recently diagnosed Type 1 diabetes mellitus. Lancet 1985;1:67–71. [21] Stiller CR, Dupre J, Gent M, Jenner MR, Keown PA, Laupacis A, et al. Effects of cyclosporine immunosuppression in insulin-dependent diabetes mellitus of recent onset. Science 1984;223:1362–7. [22] Hollander GA, Bierer BE, Burakoff SJ. Molecular and biological actions of cyclosporin A and FK506 on T cell development and function. Transfus Sci 1994;15:207–20. [23] De Filippo G, Carel JC, Boitard C, Bougneres PF. Long-term results of early cyclosporin therapy in juvenile IDDM. Diabetes 1996;45:101–4. [24] Dupre J, Stiller CR, Gent M, Donner A, von Graffenried B, Heinrichs D, et al. Clinical trials of cyclosporin in IDDM. Diabetes Care 1988;11(Suppl 1):37–44. [25] The Canadian-European Randomized Control Trial Group. Cyclosporininduced remission of IDDM after early intervention. Association of 1 yr of cyclosporin treatment with enhanced insulin secretion. The Canadian-European Randomized Control Trial Group. Diabetes 1988;37: 1574–82.
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
ARTICLE IN PRESS Available online at
ScienceDirect www.sciencedirect.com Diabetes & Metabolism xxx (2014) xxx–xxx
Short report
Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice M.S. Wong a , A. Tso a , M. Ali a , W.J. Hawthorne b , N. Manolios a,∗ a b
Rheumatology Department, The University of Sydney/Westmead Hospital, Westmead NSW 2145, Australia Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead NSW 2145, Australia Received 19 December 2013; received in revised form 25 January 2014; accepted 31 January 2014
Abstract The aim of this study was to investigate the effectiveness of immunomodulatory peptides in preventing the spontaneous onset of Type 1 diabetes in NOD mice. Two such peptides, CP and C1, were injected intraperitoneally in NOD mice three times a week starting at two different time points, nine weeks and 11 weeks of age, and blood sugar levels monitored for the development of diabetes. CP was shown to be effective in delaying the onset of diabetes compared to control (P = 0.006). The timing of peptide administration was crucial since delay in treatment did not prevent the onset of diabetes (nine weeks versus 11 weeks of age). C1 was effective in delaying the onset of Type 1 diabetes with borderline significance when given at week 11 (P = 0.05). These findings confirm the efficacy of these peptides in the prevention and possible treatment for Type 1 diabetes and thereby create new opportunities for genetic manipulation. © 2014 Elsevier Masson SAS. All rights reserved. Keywords: Immunosuppression; Non-obese diabetic (NOD) mouse; Peptide; Type 1 diabetes (T1D); Core peptide
1. Introduction Type 1 diabetes (T1D) is an autoimmune T-cell mediated disease leading to elevated blood glucose levels (BGLs). T1D is a common disease affecting 87,100 individuals in Australia, who require daily insulin injections [1]. These individuals must also monitor their BGLs, diet and exercise regularly to ensure control of their diabetes. Unfortunately, many of these patients will subsequently develop secondary complications from their disease and place an enormous personal and financial burden on the community and government. In Australia, the total annual cost (healthcare costs, the cost of carers and Commonwealth government subsidies) is $570 million, with $4669 being the average annual cost per person [2]. For individuals that have not developed secondary complications, the average annual cost is $3468, however if secondary micro- and macro-vascular complications develop, the average annual cost rises to $16,698 [2]. Several potential treatment options for patients with endstage diabetic renal failure include simultaneous pancreas and kidney transplantation; and more recently, to a subgroup of very
∗
Corresponding author. Tel.: +61 2 98458099; fax: +61 2 98458317. E-mail address: [email protected] (N. Manolios).
brittle diabetics transplantation of islets [3,4]. The problem with both of these is rejection of the graft or islets, and the secondary complications arising from the use of immunosuppressive therapy, such as prednisone, tacrolimus and cyclosporin. Newer approaches and agents such as interleukin (IL)-18 and IL-7Ra inhibitors, have being used and examined with limited success [5,6]. Core peptide (CP; GLRILLLKV) is a novel immunosuppressive peptide that has the potential to treat T1D by inhibiting the activation of T-cells, and thus the T-cell mediated destruction of islets. CP consists of two basic positively charged and hydrophilic amino acids (arginine and lysine) separated by four neutral and hydrophobic amino acids [7]. The functional significance of this peptide was published in 1997 when it was shown that CP inhibited T-cell activation in vivo and in vitro [8]. In animal studies, CP given subcutaneously significantly reduced the induction of T-cell mediated inflammation in adjuvant induced arthritis (AIA), cyclophosphamide-induced diabetes, experimental allergic encephalomyelitis (EAE) and delayed type contact hypersensitivity [8,9]. C1 ( ) is a cyclic peptide similar in sequence to CP which has alternating D-, L- amino acids, increasing the peptide’s stability and resistance to proteolytic
1262-3636/$ – see front matter © 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.diabet.2014.01.007
Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007
+Model DIABET-599; No. of Pages 6 2
ARTICLE IN PRESS M.S. Wong et al. / Diabetes & Metabolism xxx (2014) xxx–xxx
degradation [10–12]. C1 consists of two basic positively charged and hydrophilic amino acids (arginine and lysine) separated by five neutral and hydrophobic amino acids. C1 has the same ability to inhibit T-cell activation and IL-2 production as CP and in comparative studies, C1 was shown to be more effective than CP in its ability to inhibit T-cells (P < 0.012) [12]. In animal models of asthma and arthritis, C1 has been shown to be effective in suppressing the immune response [12]. This study examined the effects of CP and C1 on the onset of diabetes in non-obese diabetic (NOD) mice. This model exploits the fact that approximately 60–80% of NOD mice spontaneously develop T1D by a similar mechanism seen in human T1D [13,14] and aims to address if CP and C1 can inhibit the onset and severity of diabetes. 2. Methods 2.1. Animals All experimental protocols and methods were approved by the Western Sydney Local Health District (WSLHD) Animal Ethics Committee (AEC) and all experiments were performed in accordance with its guidelines. Female non-obese diabetic (NOD) mice were obtained at 8 weeks of age from the Animal Resources Centre (Murdoch, WA, Australia) and housed in the WSLHD Animal facility located within Westmead Hospital. Food and water was given ad libitum. The mice were randomly placed into a control group (n = 22) and treatment groups. In one group CP was given from nine weeks of age (n = 19); and in the second group CP was given from 11 weeks of age (n = 19). A third group was given C1 from 11 weeks (n = 14). Mice were deemed diabetic when the BGLs ≥ 15 mM on two consecutive readings.
can describe a situation where the vital event has not occurred; in this case, a mouse that has not yet developed diabetes. 2.5. Insulitis scoring Histological analysis was performed on pancreatic tissue collected from control and CPW9 mice at 22 weeks. A proportion of pancreatic tissue in the CPW9 group was collected three weeks after treatment ceased (at 25 weeks) and insulitis was scored separately from tissue collected at 22 weeks. The severity of insulitis was scored for at least 10 islets in each group according to Jimeno et al. [15]. When there was no lymphocytic infiltration, the sample was assigned “0, no-insulitis”. When lymphocytic infiltrations were seen around the islet and up to 30% of the islet, the sample was assigned as “1, peri-insulitis”. “2, moderate insulitis” was assigned to samples where there were lymphocytic infiltrations between 30% and 50% of the islet. Samples with lymphocytic infiltrations of more than 50% of the islet were assigned as “3, severe insulitis”. Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney tests were used to test for differences in insulitis scores and for pair-wise comparisons between groups, respectively. 3. Results As expected, 64% of the mice in the control group developed diabetes and only 36% of mice remained diabetes-free during a 13-week period of observation (week 9–22). The earliest onset
2.2. Treatment Peptides were dissolved in milliQ water and were sonicated for 30 min. The solutions were filter sterilised and stored at 4 ◦ C. CP was administered intraperitoneally (i.p.) at a concentration of 0.4 mg three times a week from either nine weeks or 11 weeks of age prior to the onset of diabetes. C1 was administered i.p. at a concentration of 0.1 mg three times a week from 11 weeks of age and the control group received no treatment. All treatments ceased at 22 weeks. 2.3. Monitoring BGLs Mice in all groups were fasted for at least 2 h prior to the measurement of their BGLs three times a week. 2.4. Kaplan-Meier survival plot A Kaplan-Meier survival plot was used to assess the percentage of NOD mice that developed diabetes over time. This statistical method is a common technique, performed to analyse survival-time data, when there is “censored” data. Censored data
Fig. 1. Kaplan-Meier survival plot comparing the number of female NOD mice that developed diabetes, with or without immunosuppressive peptide treatment. The control group (n = 22) is represented by the black solid line (A). Mice (n = 19) treated with 0.4 mg CP from 9 weeks of age is represented by the red solid line (B) and the mice (n = 19) treated with 0.4 mg CP from 11 weeks of age by the red dotted line (C). The mice (n = 14) treated with 0.1 mg C1 from 11 weeks of age is represented by the blue dotted line (D). By the end of the study, 14/22 (64%) mice in the control group had developed T1D. By contrast, only 4/19 (21%) mice developed diabetes in the group treated with CP from 9 weeks of age (B). In the group treated with CP from 11 weeks, 7/19 (37%) mice developed diabetes. Borderline significance (P = 0.05) was achieved when comparing the control group to the group treated with C1 from 11 weeks where 4/14 (29%) mice developed diabetes.
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of diabetes in the control group was at 11.4 weeks of age. By contrast in the CP treated group commencing at nine weeks of age (CPW9 ) 79% of the mice remained diabetes-free and only 21% developed diabetes (P = 0.006). In this treatment group, the onset of diabetes was delayed by four weeks compared to the control group. In the CP treated group commencing at 11 weeks
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of age (CPW11 ) 63% of the mice remained diabetes-free and 37% of the mice developed diabetes, which was not significantly different from the control group. The earliest onset of diabetes noted in this treatment group was 11.4 weeks. In mice given C1 from 11 weeks of age (C1W11 ), 71% of mice remained diabetes-free while 29% of the mice developed
Fig. 2. Blood glucose levels of mice with or without immunosuppressive peptide treatment: a: untreated mice (n = 22); b: mice given CP from nine weeks of age (CPW9 , n = 19); c: CP given from 11 weeks of age (CPW11 , n = 19); d: mice given C1 from 11 weeks of age (C1W11 , n = 14).
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diabetes (P = 0.05) during an 11-week period of observation. The earliest onset of diabetes in the C1 treatment group was delayed by one week to 12.4 weeks. These results suggest that prophylactically administered CP was able to prevent the onset of T1D in NOD mice for more than 90 days. Any delay in treatment was not effective.
The Kaplan-Meier survival plot (Fig. 1) shows the number of mice that developed diabetes after three months observation and the BGL curves for each group are shown in Fig. 2. Histology of pancreatic tissue obtained from mice at the end of the study showed that CP ameliorated the T-cell mediated destruction of islets (Fig. 3). The stacked bar curve (Fig. 4) shows the
Fig. 3. Immunohistochemical staining of pancreatic islets: a: haematoxylin and eosin stain of pancreatic tissue depicting an islet; b: insulin stain within an islet; c: haematoxylin and eosin stain of pancreatic tissue from a mouse treated with CP that did not develop diabetes, depicting an islet (lower arrow) surrounded by lymphocytes (upper arrow); d: haematoxylin and eosin stain of pancreatic tissue from a mouse treated with CP that did not develop diabetes. Despite the lymphocytic infiltration the islets are still functional as depicted by the insulin stain.
Fig. 4. Stacked bar chart comparing the distribution of insulitis scores between control and CPW9 groups. The severity of insulitis was reduced (P = 0.010) in islets from the CPW9 group (n = 11) compared to islets in the control group (n = 41). No difference in insulitis (P = 0.076) was seen in islets from the CPW9 group regardless of whether the pancreas was collected immediately or three weeks after end of treatment (n = 80).
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distribution of insulitis scores between controls and the CPW9 pancreatic tissue collected three weeks apart. There was a statistical difference in insulitis scores between the three groups (P = 0.024). In particular, the insulitis scores in the control group were significantly higher than those in the CPW9 group (P = 0.01), indicating that treatment with CP reduced the severity of lymphocytic infiltration. Although it appeared that insulitis increased when CP was withheld for the final three weeks, there was no statistical difference between insulitis scores in the CPW9 (three weeks after end of Rx) group and the CPW9 group (P = 0.076). 4. Discussion The ability to prevent the onset of diabetes and maintain euglycaemia would have dramatic and significant effects in any T1D patient if achieved. The Diabetes Control and Complications Trial and Follow-up Study (DCCT) found that maintaining normal blood glucose levels reduces the risk of secondary complications [16]. There was a 76% decreased risk of eye disease, 50% reduced risk of kidney disease and a 60% reduced risk of nerve disease. A continuation of the DCCT, the Epidemiology of Diabetes Interventions and Complications study (EDIC) found that there was a 42% decreased risk in cardiovascular disease occurrence and a 57% reduced risk of nonfatal heart attack, stroke or death from cardiovascular causes [17]. Therefore, there is a significant advantage that even delaying the onset of T1D by a few years can dramatically reduce the incidence of secondary complications. In NOD mice, by the time mice are symptomatic at 12–14 weeks, 60–90% of the islet cell mass had already been destroyed [18]. By pre-treating NOD mice with CP from nine weeks of age, the onset of T1D could be delayed. As shown histologically, CP was able to influence the T-cell response with less cellular infiltrate and inflammation. CP given at 11 weeks of age did not delay the onset of disease. By contrast, C1 may be a more powerful immunosuppressive agent than CP in preventing the progression of T1D because when given at week 11 CP had no effect, however treatment with C1 was borderline significant compared to the control group (P = 0.05). CP acts on T-cells at the transmembrane level to inhibit signal transduction following antigen recognition [7]. Published studies have indicated that CP interferes with the interactions between TCR-␣, CD3␦ and dimers [9,19]. Prevention of a completely assembled TCR-CD3- complex inhibits signal transduction and activation of T-cells. By giving CP i.p. to NOD mice at an early age, it was hypothesised that CP will inhibit antigen activation of islet-targeting T-cells. The mechanism of action for C1 has not been clearly defined but presumed to be similar to CP. A limitation of the results could be the low animal numbers (14 mice in the C1 treated group) and increasing this number may possibly lead to a more significant difference compared to controls. Furthermore, treatment of mice with C1 from the earlier time point, nine weeks of age as in the CPW9 group, could influence the results. Administering treatment to coincide with the beginning of insulitis, which begins even earlier than nine weeks
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of age, at three to four weeks of age in NOD mice [14], could dramatically improve the results and will be further investigated. Although there was a statistical significance noted between the BGLs of CP-treated and non-treated mice, further studies and longer periods of observation are needed. Subsequent studies could also include the analysis of the T-cell phenotype in the spleen and pancreatic lymph nodes of protected mice, measuring the changes in cytokine production by T-cells in the presence of CP and C1 as well as trialling C1 as an oral agent. Cyclosporin is also a cyclic peptide that is an immunosuppressant that has been used in the treatment of T1D [20,21]. Cyclosporin inhibits T-cell proliferation and the destruction of islets by blocking calcineurin and reducing IL-2 production [22]. Treatment with cyclosporin after clinical presentation of T1D has been shown to conserve insulin production, however the protective effects were not permanent [23,24]. Furthermore, the cyclosporin dose required to induce diabetes remission leads to nephrotoxicity and other adverse effects [21,25]. Development of new immunosuppressives, such as C1 and CP, could provide further drug choices in the treatment of T1D. Furthermore, the linear sequence of CP can be converted into cDNA, unlike cyclosporin, and may be used to induce tolerance when transduced into dendritic cells or provide a novel means of immunosuppression using gene therapy to transduce islets and prevent islet cell destruction. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments We express our sincere appreciation to our funding bodies – The Rebecca L. Cooper Medical Research Foundation, The Wenkart Foundation and Research Trust Fund of Rheumatology Dept., Westmead. We would also like to thank Ms Virginia James for assistance with the histology sections and Ms Karen Byth for statistical analysis. Appendix A. Supplementary data Supplementary data (French abstract) associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.diabet.2014.01.007. References [1] Australian Institute of Health and Welfare (AIHW). Diabetes prevalence in Australia: detailed estimates for 2007-08. Canberra: AIHW: 2011 Contract No.: Cat. no. CVD 56. [2] Colagiuri R. DiabCo$t type 1: assessing the burden of Type 1 diabetes in Australia. Canberra: A. C. T.: Diabetes Australia; 2009. p. 28. [3] Maffi P, Scavini M, Socci C, Piemonti L, Caldara R, Gremizzi C, et al. Risks and benefits of transplantation in the cure of Type 1 diabetes: whole pancreas versus islet transplantation. A single center study. Rev Diabet Stud 2011;8:44–50.
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Please cite this article in press as: Wong MS, et al. Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice. Diabetes Metab (2014), http://dx.doi.org/10.1016/j.diabet.2014.01.007