The Immunology of Type 1 Diabetes A. McGovern

The Immunology of Type 1 Diabetes: β-cell Murder or Suicide?by Andrew McGovern SSC Tutor: Dr Wendy Macfarlane

The causes of Type 1 diabetes (T1D) are unclear and provide continuing source of debate amongst scientists in the field of diabetes research. One of the pertinent unanswered questions is whether the beta cells are targeted for destruction or promote their own death; do they die by murder or suicide? This essay attempts to address this issue as well as proposing a novel hypothesis to explain the onset of diabetes.

IntroductionT1D makes up between 5-10% of all cases of diabetes mellitus.1 It is an autoimmune disease which involves the irreversible destruction of the insulin producing β-cells of the pancreas. The condition results in the affected individual requiring lifelong insulin replacement therapy, usually in the form of several insulin injections daily or via an insulin pump.2 Current cures focus on replacing the destroyed β-cells by either islet cell transplantation or complete pancreas transplantation in patients with other transplant indications. However, these methods often only provide a temporary cure and require a lifelong immunosuppressive drug regime which has numerous and considerable side effects.2 There is increasing pressure to identify a cure for T1D as the prevalence of the disease is increasing worldwide by around 3.0% per annum.3

T1D presents clinically when between 60-80% of the β-cell mass has been destroyed, classically with a 2-6 week history of polyuria, polydipsia and weight loss.2,4 This late presentation is the result of the existence of a pre-diabetic state, where β-cell attack is occurring and there is localised inflammation of the pancreatic islets (insulitis), but where the individual has no clinical signs of diabetes. This is demonstrated in Figure 1.5If the pre-diabetic state could be identified and the pathology reversed it may be possible to prevent the clinical

1

Figure 1. Stages in the development of T1D.5

The Immunology of Type 1 Diabetes A. McGovern

symptoms of T1D from occurring. Therefore, an understanding of the aetiology of T1D may provide a number of potential methods of intervention in individuals with an identified pre-diabetic state. It may also reveal possible approaches to prevention of the disease such as vaccination or targeted immunosuppression.

The causes of T1DCurrently a number of causes of T1D have been identified or suggested, although there is no consensus as to which of these is the most important causal factor and no single factor has been identified which is always associated with T1D. Epidemiologic studies suggest possible contributions from genetic factors, pathogens (primarily viral), cow’s milk, nitrosamines, zinc, vitamin D and parental age.6 Significant differences exist in concordance of T1D prevalence between monozygotic (7%) and dizygotic twins (43%), demonstrating a strong genetic liability.7 Further evidence suggests that genetic susceptibility to T1D is polygenic, with 40-50% of this component accounted for by the presence of particular HLA alleles.8 These include the HLA class II alleles HLA-DRB1-DQB1 and HLA-DRB1-DQA1.9 HLA class II proteins are involved in the presentation of protein fragments as a result of phagocytosis by antigen presenting cells, and are consequently important modulators of the adaptive immune defence. In addition to the HLA loci, more than 20 further genome loci have been identified which may provide supplemental T1D susceptibility.2,9

There is increasing evidence to suggest a causal role of viral pathogens in the onset of T1D in humans. Associations with T1D has been found with a number of enteroviruses, rubella, mumps, rotavirus and cytomegalovirus (CMV).1 It has been demonstrated that the Coxsackie B virus (an enterovirus) is present in a high proportion of patients at the onset of T1D.10 It has also been established that 15% of patients newly diagnosed with T1D have identifiable CMV genome in their sera.11 However other studies have suggested any causal relationship is minor with only 1.5% of children, with an identified congenital CMV infection, developing T1D.12

A number of mechanisms by which viral infections induce the destruction of β-cells have been suggested:1

1. Infection of pancreatic β-cells by pathogen may directly induce cell lysis.

2. Infection of β-cells by pathogen may result in the expression of viral antigens and destruction by cell mediated immunity.

3. Viral infection may induce bystander activation of autoreactive lymphocytes.

4. Release or expression of viral superantigens may induce polyclonal expansion of lymphocytes. This could include autoreactive cells.

5. Successive infections by two viruses may precipitate autoimmunity.

This latter postulated mechanism forms the basis of the novel hypothesis for the onset of insulitis and T1D suggested here. These mechanistic suggestions

2

The Immunology of Type 1 Diabetes A. McGovern

implicate the role of the immune system in the destruction of the β-cell, and are in agreement with a β-cell murder hypothesis.

Nutritional factors implicated in the onset of T1D include the early introduction of cows milk to the diet. A meta analysis identified a 1.6 fold increase in the risk of developing T1D in infants exposed to cows milk before 4 months.13 However it is not clear whether this is a result of early exposure to cows milk antigens or due to early withdrawal from the immunoglobulin mediated protection provided through breastfeeding.6 High levels of nitrosamines have also been suggested as a potential cause of T1D. Diets containing a high intake of smoked and cured mutton show a correlation with T1D occurrence.6 Smoked and cured meats contain high levels of nitrosamine compounds. It has been suggested that these compounds have a damaging effect on the development of β-cells during the embryonic period.6

So far there very little evidence has been proposed here to support the suicide hypothesis of β-cell destruction. This evidence arises from examination of the mechanism of apoptosis that occur in insulitis. It has been demonstrated that the majority of β-cells die via the apoptotic pathway.14,15 This is initiated by the interaction of Fas and Fas-ligand, perforin and granzyme, and tumor necrosis factor.14 Non-obese diabetic mouse research has identified the upregulation of Fas expression on β-cells as a causative factor for β-cell destruction,16 thereby implying a degree of β-cell suicide. However, some evidence suggests that upregulation of Fas expression is induced by inflammatory mediators (specifically interleukin 1β and interferon-γ).17 Hence suggesting that Fas expression is an intermediate step in the process of β-cell murder and an unavoidable outcome of insulitis, however the evidence is inconclusive.

This demonstrates that evidence can be provided to support both a murder and suicide hypothesis. However either view is probably an over simplification of a complex condition. As, currently no single cause has been identified in the initiation of insulitis and subsequent T1D, and It also appears unlikely that a single causative agent will be identified, we must therefore consider a multifactorial aetiology.

A novel view of T1D onsetProposed here is the novel theory of insulitis threshold. In this theory a succession of environmental triggers is required to initialise the onset of a pre-diabetic state. That is to say, several factors are required to prime the immune system for insulitis followed by exposure to an activating factor. It has previously been proposed that multiple viral infections hasten disease onset once an individual has entered the pre-diabetic state.18 However this is a more general model suggesting a broader role, in the onset of pre-diabetes, of multiple factors (which will be subsequently referred to as diabetogens). Likely diabetogens include viral infections, which increase the number of autoreactive lymphocytes through molecular mimicry and bystander activation.1 It has been demonstrated that molecular mimicry by lymphocytic-choriomenigitis virus in the rat insulin promoter-lymphocytic-choriomenigitis virus nucleoprotein (RIP-LCMV-NP) transgenic mouse model of autoimmune diabetes leads to the production of autoreactive T cells.18 These viral mimics were shown to produce an acceleration of insulitis in the pre-diabetic state but not to initiate a sufficient number of lymphocytes to cause diabetes in the naïve lymphocyte populations of unprimed animals even when insulitis was temporarily initiated.18

3

insulitis

The Immunology of Type 1 Diabetes A. McGovern

It was also demonstrated that central tolerance mechanisms (thymic selection) were capable of removing autoreactive T cells.18 Further work suggests that despite thymic selection, a significant number of high affinity T cells remain and are activated on repeated infection.1

In humans infection by a number of these diabetogenic viruses may produce a number of autoreactive T cell populations, via molecular mimicry and other mechanisms. It is likely that a considerable number of these autoreactive cell populations will remain dormant in the substantial lymphatic network surrounding the pancreas. It has been previously suggested that autoreactive clones are capable of remaining silent in the lymph nodes where they evade regulatory mechanisms.19 These combined factors would push the individual towards the insulitis threshold as shown in Figure 2. A minor trigger would then be needed to recruit the autoreactive T cells to the islets and initiate the irreversible pre-diabetic state. Likely triggers include pathogen derived superantigens, the lowering of insulitis threshold by neuronally induced inflammation or simply pathogen induced insulitis and bystander activation.1,20

4

0 2 4 6 8 10 12 14Age (years)

Aut

orea

ctiv

ity

insulitis threshold

Figure 2. Two individuals challenged with the identical diabetogenic factors (marked with arrows). Subject A (blue) has genetic susceptibility to diabetes, subject B (red) is normal. At age 14 subject A enters a prediabetic state, whereas subject B remains far below the insulitis threshold. Note at age 11 subject A gets insulitis but does not cross the insulitis threshold and enter the pre-diabetic state. This is in agreement with mouse models.18

Autoreactivity is a function of the number of autoreactive lymphocytes, their proximity to the islets and their responsiveness to activation, amongst other factors.

The Immunology of Type 1 Diabetes A. McGovern

An insulitis threshold theory may also explain the discrepancies between studies looking at the relationship between T1D and CMV. Studies describing T1D onset following an early or congenital CMV infection identify only a weak link,12,21 compared to those which look at CMV infection in newly diagnosed individuals.1,11 This suggests that CMV is a diabetogen in early childhood but requires additional diabetogenic triggers to exceed the insulitis threshold. However if infection occurs after a number of diabetogenic triggers have already challenged an individual then CMV acts as more potent diabetogen by amplifying the remnant effects of the previous diabetogens. Thus a CMV infection occurring later in life would be a more potent diabetogen as it likely that the individual has been exposed to other diabetogens during their lifetime. Closer inspection of the hypothesised mechanisms by which CMV acts as a diabetogen also provides support for this theory. It has been demonstrated that CD4+ T cells that recognise the antigen presented CMV protein UL57 also respond to autoantigen.22 This suggest a role of molecular mimicry which explains the weakly diabetogenic role of CMV in congenital infection. CMV has also been implicated in the proliferation of polyclonal T cell populations through both bystander activation and production of superantigen.1,23 This provides an explanation for it’s increased diabetogenic effects in later infection, as polyclonal T cell proliferation is likely to include autoreactive cells remaining from previous diabetogen encounters. Additionally, this prediction provides parameters which are testable in animal models: Altering the order of infection with known diabetogenic viruses will produce varying prevalences of diabetes onset, however large sample sizes would be needed to confirm this with statistical significance as the difference may be slight.

Other identified factors may act to lower the insulitis threshold such a neuronally induced release of inflammatory mediators or systemic infection. Such neuronal mechanisms have been identified that require the release of inflammatory molecules for normal physiological functioning of islet cells.20

Once the insulitis threshold has been exceeded there will be rapid proliferation of autoreactive lymphocytes and recruitment of additional immune cells contributing to insulitis. Development of autoantibodies will also result when peripheral tolerance mechanisms to β-cells are lost as a result of the localised inflammation.

Genetic effects can also be accounted for by the insulitis threshold theory. Particular alleles, such as the HLA-DRB1-DQB1 and HLA-DRB1-DQA1 protein types, may confer a susceptibility to particular diabetogens. Individuals without these susceptibly alleles are therefore immune to the effects of these diabetogens and less likely to cross the insulitis threshold. Challenging any individual with a large number of general diabetogens will still induce diabetes.

Whilst this theory provides an explanation for the links between known diabetogenic factors and the incidence of T1D onset, it is not complete. It explains that exposure to a single diabetogen, produces insufficient autoreactive lymphocytes to initiate or sustain insulitis, but will prime the individual to exceed the insulitis threshold on exposure to a subsequent diabetogen. However, no explanation for the decreased risk of T1D onset in adults is evident at present. Additional insight into the variation between adult and child tolerance mechanisms may provide an explanation. An analysis of the full complement of diabetogenic factors may also explain this pattern.

Conclusion

5

The Immunology of Type 1 Diabetes A. McGovern

Diabetes has been demonstrated to be caused by a number of factors both genetic and environmental. Theories about diabetes onset should consider all these factors and one such theory has been suggested here. Emerging evidence for these causal factors is making the consideration of β-cell suicide appear increasing less likely. However the role and mechanisms of FAS death receptor expression has not yet been fully elucidated and are vital in clarifying this distinction.

Word count: 2022

In t

References1. Werf Nvd, Kroese FGM, Rozing J, Hillebrands J-L. Viral infections as potential

triggers of type 1 diabetes. Diabetes/Metabolism Research and Reviews. 2007;23:169-83.

2. Kumar P, Clark M. Clinical Medicine. 5th ed. London: W. B. Saunders; 2002.3. Onkamo P, Väänänen S, Karvonen M, Tuomilehto J. Worldwide increase in

incidence of Type I diabetes--the analysis of the data on published incidence trends. Diabetologia. 1999;42(12):1395-403.

4. Greiner DL, Rossini AA, Mordes JP. Translating Data from Animal Models into Methods for Preventing Human Autoimmune Diabetes Mellitus: Caveat Emptor and Primum non Nocere. Clinical Immunology. 2002;100(2):134-43.

5. Eisenbarth G. Type I diabetes mellitus. A chronic autoimmune disease. The New England Journal of Medicine. 1986;324(21):1360-8.

6. Fronzo RAD, Ferrannini E, Keen H, Zimmet P, editors. International Textbook of Diabetes Mellitus. 3rd ed. UK: John Wiley & Sons, Ltd.; 2004.

7. Hyttinen V, Kaprio J, Kinnunen L, Koskenvuo M, Tuomilehto J. Genetic Liability of Type 1 Diabetes and the Onset Age Among 22,650 Young Finnish Twin Pairs. Diabetes Research and Clinical Practice. 2003;52:1052-5.

8. Field L. Genetic linkage and association studies of Type I diabetes: challenges and rewards. Diabetologia. 2002;45(1):21-35.

9. Spielman R, Baker L, Zmijewski C. Gene dosage and susceptibility to insulin-dependent diabetes. Annals of Human Genetics. 1980;44(2):135-50.

10. Clements G, Galbraith D, Taylor K. Coxsackie B virus infection and onset of childhood diabetes. Lancet. 1995;346(8969):221-3.

11. Pak CY, Cha CY, Rajotte RV, McArthur RG, Yoon JW. Human pancreatic islet cell specific 38 kilodalton autoantigen identified by cytomegalovirus-induced monoclonal islet cell autoantibody. Diabetologia. 1990;33(9):569-72.

6

The Immunology of Type 1 Diabetes A. McGovern

12. Ivarsson S, Lindberg B, Nilsson K, Ahlfors K, Svanberg L. The prevalence of type 1 diabetes mellitus at follow-up of Swedish infants congenitally infected with cytomegalovirus. Diabetic Medicine. 1993;10(6):521-3.

13. Gerstein H. Cow's milk exposure and type I diabetes mellitus. A critical overview of the clinical literature. Diabetes Care. 1994;17(1):13-9.

14. Pearl-Yafe M, Yolcu ES, Yaniv I, Stein J, Shirwan H, Askenasy N. The dual role of Fas-ligand as an injury effector and defense strategy in diabetes and islet transplantation. BioEssays. 2006;28(2):211-22.

15. Bach J. Organ-specific autoimmunity. Immunology Today. 1995;16(7):353-5.16. Silva D, Petrovsky N, Socha L, Slattery R, Gatenby P, Charlton B. Mechanisms of

accelerated immune-mediated diabetes resulting from islet beta cell expression of a Fas ligand transgene. Journal of Immunology. 2003;170(10):4996-5002.

17. Thomas H, Darwiche R, Corbett J, Kay T. Evidence that beta cell death in the nonobese diabetic mouse is Fas independent. Journal of Immunology. 1999;163(3):1562-9.

18. Christen U, Edelmann KH, McGavern DB, Wolfe T, Coon B, Teague MK, et al. A viral epitope that mimics a self antigen can accelerate but not initiate autoimmune diabetes. The Journal of Clinical Investigation. 2004;114(9):1290-8.

19. Pearl-Yafe M, Iskovich S, Kaminitz A, Stein J, Yaniv I, Askenasy N. Does physiological beta cell turnover initiate autoimmune diabetes in the regional lymph nodes? Autoimmune Reviews. 2006;5(5):338-43.

20. Tsui H, Razavi R, Chan Y, Yantha J, Dosch H. 'Sensing' autoimmunity in type 1 diabetes. Trends in Molecular Medicine. 2007;13(10):405-13.

21. Hiltunen M, Hyöty H, Karjalainen J, Leinikki P, Knip M, Lounamaa R, et al. Serological evaluation of the role of cytomegalovirus in the pathogenesis of IDDM: a prospective study. Diabetologia. 1995;38(6):705-10.

22. Hiemstra HS, Schloot NC, Veelen PAv, Willemen SJM, Franken KLMC, Rood JJv, et al. Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(7):3988-91.

23. Dobrescu D, Ursea B, Pope M, Asch A, Posnett D. Enhanced HIV-1 replication in V beta 12 T cells due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell. 1995;82(5):753-63.

7