**5. Enterovirus and type 1 diabetes: Experimental approach**

In previous sections of this review, clinical studies that were conducted to assess the rela‐ tionship between enteroviruses and T1D have been presented. A significant association be‐ tween enterovirus infection and T1D, particularly for studies that used molecular methods, has been displayed, and when identified the most often involved enteroviruses were cox‐ sackieviruses B. Experimental studies have been performed to understand the possible link between enterovirus and T1D. In the present section, the results of in vivo studies on one hand and those of in vitro studies on the other hand are presented and analyzed.

### **5.1. In vivo studies in animal models**

**Biological samples**

46 Type 1 Diabetes

PBMC plasma stools throat

**Number of Cases/ Controls**

10/20 10/20 10/20 10/20 **Children/ adults patients**

PBMC 24/24 24/0 18/7

leucocytes 112/56 112/0 93/4

Pancreas 149/21 NI 0/7

Pancreas 65/40 0/65 4/0

Pancreas 72/39 NI 44/3

Intestine 12/10 0/12 6/0

Pancreas 6/26 2/4 3/0 IHC

**Positives cases/ Controls p value**

p\*

p\*

2/0 1/0 0/0

p\*

p\*

p<0.001

p=0.015 9/1 p=0.004

Immunohistochemistry, HIS: Hybridization in situ, NI: Not Indicated, p\*: p value not mentioned.

**5. Enterovirus and type 1 diabetes: Experimental approach**

hand and those of in vitro studies on the other hand are presented and analyzed.

10/0 4/0 p>0.05

**Methods of detection**

Electronic microscope Cell culture RT-PCR

HIS IHC

**Table 3.** Detection of enterovirus and/or their components (RNA, proteins) in biological samples of patients with type 1 diabetes. PBMC: Peripheral Blood Mononuclear Cells, RT-PCR: Retrotranscription Polymerase Chain Reaction, IHC:

In previous sections of this review, clinical studies that were conducted to assess the rela‐ tionship between enteroviruses and T1D have been presented. A significant association be‐ tween enterovirus infection and T1D, particularly for studies that used molecular methods, has been displayed, and when identified the most often involved enteroviruses were cox‐ sackieviruses B. Experimental studies have been performed to understand the possible link between enterovirus and T1D. In the present section, the results of in vivo studies on one

**Reference country**

RT-PCR Yin et al., 2002 Sweden

RT-PCR Toniolo et al., 2010 Italy

RT-PCR Schulte et al., 2010 Netherlands

IHC Foulis et al., 1990 England

HIS Ylipaasto et al., 2004 Finland

IHC Richardson et al., 2009 England

Dotta et al., 2007 Italy

Oikarinen et al., 2007 Finland

In order to analyse the hypothesis that enterovirus infections enhance or elicit autoimmune disorders such as T1D, a significant body of evidence is derived from investigations using animal models. Most of them used to explore research hypotheses regarding the relation‐ ship between enteroviruses and type 1 diabetes are mouse models (NOD, C57BL/k, C57BL/6, SJL/J, DBA/2, SWR/J, BALB/c, B10, CD-1…) [83]. Despite their limitation in diseas‐ es investigations, experimental models have greatly contributed to our knowledge of human diseases. The predominance of murine models for the investigation of the relationship be‐ tween enteroviruses and T1D is due, among others, to a physiology relatively similar to that of human beings and the presence of specific receptors, the more prominent of them could be the coxsackievirus and adenovirus receptor (CAR) which is a receptor for coxsackievirus B [86]. Therefore experimental datas have been obtained from models based on infection with coxsackievirus B (CVB) (figure 3).

#### *5.1.1. Enterovirus and immune system*

Experimental in vivo studies have contributed improving our understanding of genetic and immunological implications, enteroviral tropism and mechanisms of pancreatic β-cells de‐ struction in the context of enteroviral infection [83]. Enteroviruses generally infect the exo‐ crine pancreas, but some strains preferentially infect islets. Some studies have addressed the role of CAR, the main receptor for CVB entry into host cell, in enteroviral tropism and target organ infection. CAR is expressed by intestine, pancreas and heart epithelial cells, as well as cardiomyocytes [54]. In transgenic mice CVB3 titers were markedly reduced in CAR-defi‐ cient tissues and pancreatic CAR deletion induced a strong attenuation of pancreatic CVB3 infection and pancreatitis [86].

The development of innate and adaptive immune responses is mediated by type I interfer‐ ons (IFNs) produced early during viral infection to induce an antiviral state within target cells. Experimental studies have shown that mice deficient in type I IFNs receptor are more susceptible and die more rapidly than controls when infected with CVB3 [169, 40]. An effi‐ cient immune response depends on rapid recognition of viruses by the innate immune sys‐ tem and this recognition is primarily achieved by pattern-recognition receptors such as tolllike receptors (TLRs), retinoid-inducible gene 1-like receptors (RIG-1) and NOD-like receptors. It is noteworthy that interactions between NOD-like receptors and enteroviruses are still poorly understood.

Toll-like receptors are transmembrane glycoproteins expressed on the cytoplasmic mem‐ brane or in intracellular vesicals of several immune and non-immune cell populations; while RIG-I-like receptors, represented by RIG-I and the interferon-induced with helicase C do‐ main 1 (IFIH-1), also called melanoma differentiation-associated gene 5 (MDA5) are ex‐ pressed in the cytosol of most cell types [91]. Among TLRs, TLR3, known to be doublestranded RNA sensor on monocytes, is known to be crucial for the survival of mice infected with CVB4 [123]; and the production of cytokines by murine plasmocytoid dendritic cells have been shown to be closely linked with CVB detection and recognition by TLR7 [168]. The MDA5 is in turn essential for type I IFNs responses to CVB, since MDA5 knockout mice are deficient to type I IFN and are prone to early death when infected with CVB (Wang et al., 2010). Thus, pattern-recognition receptors activation by enteroviruses results on IFNs and chemokines production which could lead to an inflammatory state in infected tissues. Moreover, these inflammatory factors enhance the overexpression of MHC-I molecules, which could result in an increased exposure of infected cells to the immune system and could initiate an autoimmune process that could directly contribute to islet cells damage [173]. However an activation of MDA-5 with any other factor can not initiate autoimmunity, whereas IFN-I-induced MDA-5 accelerated a preexistent autoimmune process in an animal model [38].

ed by bystander activation of T cells [73], which would tend to confirm early findings that have shown that infection of normal mice with CVB4 causes an overt diabetes associated

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

http://dx.doi.org/10.5772/52087

49

The mechanism behind this β-cell destruction has been explored in some studies. Analysis of the results from these studies reveals that the spontaneous development of diabetes in NOD mice can be accelerated by CVB4 infection though a "bystander" effect only if a suffi‐ cient number of pre-existing autoreactive T-cells was already present [134]. This observation was in agreement with another study which has shown that the overexpression of a TCR transgene specific to an islet autoantigen has induced diabetes onset 2-4 weeks after CVB4 inoculation in mice that do not develop diabetes spontaneously [73]. Islet cell destruction by autoreactive T-cells was the result of the release of sequestered islet antigens which followed β-cell inflammation and destruction caused by CVB infection [73, 2001]. Other studies have stated that β-cells are phagocyted by antigen-presenting cells like macrophages, rather than directly destroyed by a CVB-induced process [75, 133], because antigen-presenting cells iso‐ lated from CVB4-infected mice can induce diabetes if inoculated to non-infected mice [75]. Among T1D animal models, the NOD mouse remains far the most used and studied model. The NOD mice are susceptible to spontaneous T1D that develops over several weeks and share most aspects of human T1D [83]. In NOD mice, the disease occurs after T-cell-mediat‐ ed destruction of β-cells [87, 170]. Some studies have revealed that CVB infection effects in NOD mice appear to be contingent upon the precise moment at which infection occurs [134, 156]. Thus, rapid T1D induction can be obtained when older NOD mice are inoculated with CVB and the disease occurs much more rapidly when mice islets are already developing au‐ toimmune insulitis and high islet cells lytic viral replication are observed when à virulent strain is inoculated [156]. These findings suggest that CVB replicate more readily in aged NOD mice islet cells, especially if there is inflammation, than in those of younger animals. Another factor seems to be the magnitude of effects of CVB4 infection onto β-cells, depend‐ ing on the permissiveness of target cells, which is closely related to their sensitivity to IFNs. Indeed, coxsackievirus B4-infected-NOD mice which had defective IFNs responses have de‐ veloped an acute form of type 1 diabetes, similar to the one in humans following severe en‐ teroviral infection. Interferons act by inducing an antiviral state in target cells, including pancreatic β-cells, by reducing their permissiveness to viral entry and replication. The effect of IFNs is transmitted as an intracellular signal through the Jack-STAT signaling pathway [140]. In transgenic NOD mice that express the suppressor of cytokine signaling 1 (SOCS-1), a negative regulator of IFN action which inhibit the Jack-STAT signaling pathway, CVB4 in‐ fection has resulted in β-cell loss and diabetes onset. Similar results have been obtained dur‐ ing the same study in transgenic NOD mice of which β-cells were lacking IFN receptors. In addition to inducing on β-cells a lower permissiveness to CVB4 infection, IFNs contributed

with low insulin levels consistent with islet cells destruction [33].

also to deeply decrease their sensitivity to NK cell-mediated destruction [50].

Glutamic acid decarboxylase 65kD (GAD65), a candidate autoantigen in the pathogenesis of T1D, is expressed in pancreatic β-cells. Some findings from mice have shown that CTL (cyto‐

*5.1.3. Molecular mimicry hypothesis*

Some studies in animals have highlighted the potent role of antibodies and immune cells during enteroviral infection. Results on mice have shown that gammaglobulins are essential in limiting the scope and severity of enteroviral infection by preventing viral persistence in infected tissues [103] and T lymphocytes can deeply limit virus replication in CVB3-induced myocarditis and pancreatitis [63].

#### *5.1.2. Enteroviruses can induce diabetes in mice*

Experiments have been conducted to evaluate the ability of CVB4 to elicit diabetes in mice. These studies have shown that the pancreas was a predominant site of virus replication and the target of a strong immune response.

A CVB4 strain isolated by Yoon et al. from the pancreas of a 10-year-old boy who died of diabetic ketoacidosis and called CVB4E2, have induced hyperglycaemia with inflammation of the Langerhans islets and β-cell necrosis when inoculated to susceptible mice SJL/J [175]. A similar result was obtain in the same SJL/J mice strain when inoculated with a CVB5 strain isolated from stools of a diabetic patient [121]. In another study, CVB4E2 has led to hyperglycaemia and the appearance of anti-GAD antibodies in the vast majority of mice, suggesting a potent role of enteroviruses in initiating or accelerating autoimmunity against β-cells [57]. Diabetes with viral replication in β-cells has been also obtained when CVB4 JVB strain was inoculated to susceptible mice [174]. In addition, diabetes has been obtained in mice infected by CVB3 and CVB5 when animals were first treated with sub-diabetogenic doses of streptozotocin, a highly specific β-cell toxin. Findings from that study have re‐ vealed that virus-induced diabetes can be facilitated by cumulative effects induced by genet‐ ic factors or environmental insults (chemicals, drugs, toxins), since CVB strains (B3 and B5) used in that study ordinarily produce little if any β-cell damage [153]. Furthermore, CVB4 induced abnormal thymic, splenic and peripheral lymphocytes repertoire maturation has been described in mice and these lymphocyte maturation disorders have preceded the onset of hyperglycemia in animals [22].

In a study, CD-1 mice have been infected with the diabetogenic strain CVB4E2 and followed during one year. Results from this study have revealed a prolonged presence of viral RNA in pancreas tissue, a significant decrease in insulin levels and islets cells destruction by two mechanisms: directly by cytotoxic effects of IFN-γ-stimulated peritoneal macrophages and by an antibody-dependent mechanism through islet cell autoantibody (ICA) [133]. In anoth‐ er study, infection of mice with CVB4 has led to a rapid development of the disease mediat‐ ed by bystander activation of T cells [73], which would tend to confirm early findings that have shown that infection of normal mice with CVB4 causes an overt diabetes associated with low insulin levels consistent with islet cells destruction [33].

The mechanism behind this β-cell destruction has been explored in some studies. Analysis of the results from these studies reveals that the spontaneous development of diabetes in NOD mice can be accelerated by CVB4 infection though a "bystander" effect only if a suffi‐ cient number of pre-existing autoreactive T-cells was already present [134]. This observation was in agreement with another study which has shown that the overexpression of a TCR transgene specific to an islet autoantigen has induced diabetes onset 2-4 weeks after CVB4 inoculation in mice that do not develop diabetes spontaneously [73]. Islet cell destruction by autoreactive T-cells was the result of the release of sequestered islet antigens which followed β-cell inflammation and destruction caused by CVB infection [73, 2001]. Other studies have stated that β-cells are phagocyted by antigen-presenting cells like macrophages, rather than directly destroyed by a CVB-induced process [75, 133], because antigen-presenting cells iso‐ lated from CVB4-infected mice can induce diabetes if inoculated to non-infected mice [75].

Among T1D animal models, the NOD mouse remains far the most used and studied model. The NOD mice are susceptible to spontaneous T1D that develops over several weeks and share most aspects of human T1D [83]. In NOD mice, the disease occurs after T-cell-mediat‐ ed destruction of β-cells [87, 170]. Some studies have revealed that CVB infection effects in NOD mice appear to be contingent upon the precise moment at which infection occurs [134, 156]. Thus, rapid T1D induction can be obtained when older NOD mice are inoculated with CVB and the disease occurs much more rapidly when mice islets are already developing au‐ toimmune insulitis and high islet cells lytic viral replication are observed when à virulent strain is inoculated [156]. These findings suggest that CVB replicate more readily in aged NOD mice islet cells, especially if there is inflammation, than in those of younger animals.

Another factor seems to be the magnitude of effects of CVB4 infection onto β-cells, depend‐ ing on the permissiveness of target cells, which is closely related to their sensitivity to IFNs. Indeed, coxsackievirus B4-infected-NOD mice which had defective IFNs responses have de‐ veloped an acute form of type 1 diabetes, similar to the one in humans following severe en‐ teroviral infection. Interferons act by inducing an antiviral state in target cells, including pancreatic β-cells, by reducing their permissiveness to viral entry and replication. The effect of IFNs is transmitted as an intracellular signal through the Jack-STAT signaling pathway [140]. In transgenic NOD mice that express the suppressor of cytokine signaling 1 (SOCS-1), a negative regulator of IFN action which inhibit the Jack-STAT signaling pathway, CVB4 in‐ fection has resulted in β-cell loss and diabetes onset. Similar results have been obtained dur‐ ing the same study in transgenic NOD mice of which β-cells were lacking IFN receptors. In addition to inducing on β-cells a lower permissiveness to CVB4 infection, IFNs contributed also to deeply decrease their sensitivity to NK cell-mediated destruction [50].

#### *5.1.3. Molecular mimicry hypothesis*

are deficient to type I IFN and are prone to early death when infected with CVB (Wang et al., 2010). Thus, pattern-recognition receptors activation by enteroviruses results on IFNs and chemokines production which could lead to an inflammatory state in infected tissues. Moreover, these inflammatory factors enhance the overexpression of MHC-I molecules, which could result in an increased exposure of infected cells to the immune system and could initiate an autoimmune process that could directly contribute to islet cells damage [173]. However an activation of MDA-5 with any other factor can not initiate autoimmunity, whereas IFN-I-induced MDA-5 accelerated a preexistent autoimmune process in an animal

Some studies in animals have highlighted the potent role of antibodies and immune cells during enteroviral infection. Results on mice have shown that gammaglobulins are essential in limiting the scope and severity of enteroviral infection by preventing viral persistence in infected tissues [103] and T lymphocytes can deeply limit virus replication in CVB3-induced

Experiments have been conducted to evaluate the ability of CVB4 to elicit diabetes in mice. These studies have shown that the pancreas was a predominant site of virus replication and

A CVB4 strain isolated by Yoon et al. from the pancreas of a 10-year-old boy who died of diabetic ketoacidosis and called CVB4E2, have induced hyperglycaemia with inflammation of the Langerhans islets and β-cell necrosis when inoculated to susceptible mice SJL/J [175]. A similar result was obtain in the same SJL/J mice strain when inoculated with a CVB5 strain isolated from stools of a diabetic patient [121]. In another study, CVB4E2 has led to hyperglycaemia and the appearance of anti-GAD antibodies in the vast majority of mice, suggesting a potent role of enteroviruses in initiating or accelerating autoimmunity against β-cells [57]. Diabetes with viral replication in β-cells has been also obtained when CVB4 JVB strain was inoculated to susceptible mice [174]. In addition, diabetes has been obtained in mice infected by CVB3 and CVB5 when animals were first treated with sub-diabetogenic doses of streptozotocin, a highly specific β-cell toxin. Findings from that study have re‐ vealed that virus-induced diabetes can be facilitated by cumulative effects induced by genet‐ ic factors or environmental insults (chemicals, drugs, toxins), since CVB strains (B3 and B5) used in that study ordinarily produce little if any β-cell damage [153]. Furthermore, CVB4 induced abnormal thymic, splenic and peripheral lymphocytes repertoire maturation has been described in mice and these lymphocyte maturation disorders have preceded the onset

In a study, CD-1 mice have been infected with the diabetogenic strain CVB4E2 and followed during one year. Results from this study have revealed a prolonged presence of viral RNA in pancreas tissue, a significant decrease in insulin levels and islets cells destruction by two mechanisms: directly by cytotoxic effects of IFN-γ-stimulated peritoneal macrophages and by an antibody-dependent mechanism through islet cell autoantibody (ICA) [133]. In anoth‐ er study, infection of mice with CVB4 has led to a rapid development of the disease mediat‐

model [38].

48 Type 1 Diabetes

myocarditis and pancreatitis [63].

*5.1.2. Enteroviruses can induce diabetes in mice*

the target of a strong immune response.

of hyperglycemia in animals [22].

Glutamic acid decarboxylase 65kD (GAD65), a candidate autoantigen in the pathogenesis of T1D, is expressed in pancreatic β-cells. Some findings from mice have shown that CTL (cyto‐ toxic T lymphocytes) are cytotoxic to islet cells [44] and that T cell responses to GAD65 were detectable in prediabetic NOD mice spleens prior to disease onset [89, 152]. One of the mechanisms proposed to explain enterovirus-induced autoimmunity in T1D model is based on the cross-reactivity between CVB antigens and β-cell endogenous proteins through mo‐ lecular mimicry. Pancreatic β-cells infection by CVB will be followed by inflammatory re‐ sponse resulting in β-cell destruction and increased self-antigen presentation due to their phagocytosis by antigen-presenting cells (APCs). Since P2-C protein sequence of CVB parti‐ ally resembles that of human GAD65, both autoreactive and antiviral T-cells activated upon CVB infection, might act as strong enhancers that may accelerate or aggravate the ongoing autoimmune process [28, 151].

*5.2.1. Enterovirus infection of β-cells*

reactive) CD4+ TH1 cells [20].

kines in a rat insulinoma β-cell line (INS-1) [107].

Persistent infection of human pancreatic islets by CVB associated with alpha interferon (IFN) synthesis was observed [23]. In this study conducted by our team, human pancreatic islets obtained from adult brain-dead donors and cultured in noncoated membrane inserts were infected with CVB3 and a diabetogenic (CVB4 E2) and a non-diabetogenic (CVB4 JVB) strain of CVB4. It was displayed that both α and β cells in human pancreatic islet can be per‐ sistently infected and long term CVB replication has been observed through the presence of infectious particles in culture supernatant fluids and intracellular viral negative-strand RNA up to 30 days post infection. This study showed that human islets challenged with CVB can synthesize IFN-α which is produced by infected β-cells only. These data support the hy‐ pothesis of a role of CVB in the high levels of type I IFNs that have been detected in pan‐ creas or islets of patients with T1D [51, 76]. The viral persistence accompanied by synthesis of INF-α can enhance autoimmune processes leading to diabetes onset. The possibility that IFN-α could take part in T1D onset in genetically predisposed host have been tested in transgenic mice of which β-cells express this cytokine. It revealed that IFN-α was able to provoke the onset of the disease in transgenic animals, and that neutralizing IFN-α prevent‐ ed inflammation and diabetes [142]. The expression of IFN-α in β-cells may lead to the de‐ velopment of diabetes in transgenic mice through the activation of autoimmune (islet-

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

http://dx.doi.org/10.5772/52087

51

Recent findings have shown that type I IFNs production can be induced in CVB infected hu‐ man islet cells by intracellular viral RNA sensors such as TLR3, MDA-5 and RIG-1 genes [77]. These pattern-recognition receptors have also been told to upregulate the synthesis and production of chemokines. The sustaining of this process - IFNs and chemokines production – could be deleterious and involved in the development of autoimmunity, especially since

The infection of β-cells with CVB and the molecular pathways leading to CVB-induced βcell death have been investigated. One study was aimed to evaluate the effects of different CVB4 strains on islets morphology and insulin release and another one compared inflamma‐ tory-related genes expression in CVB4-infected and uninfected isolated human islets. Re‐ sults from these studies have revealed that even though the outcome of the infection differed, islet cells can be infected by all CVB4 strains. However, significant differences in viral titers and cell morphology were observed according to the phenotype of the strain: one with no cytopathic effect despite high virus titres (VD2921 stain), and the other with a pro‐ nounced cytopathic effect (V89-4557 strain), whereas a third one (JVB strain) have induced a significant increase of insulin release [55]. A microarray analysis of RNA from CVB4-infect‐ ed human islets have shown specific induction of several inflammatory genes, some of them encoding proteins with potent biological activity such as IL-1β, IL-6, IL-8, MCP-1 and RANTES [117]. Recently, it has been reported that, except CVB1 and CVB3, all other CVB viruses induced a dose-dependent production of pro-inflammatory cytokines and chemo‐

The release of proinflammatory cytokines may strongly contribute to maintain a local pan‐ creatic-islet inflammation that could result in an amplification of the immune attack against

persistent infection of islets cells in vitro by some CVB strains has been reported [23].

Regardless T-cells cross reactivity effects, experiments on CVB4-infected NOD mice have provided the evidence that the release of β-cell antigens followed by their presentation by APCs (antigen presentation cells) such as macrophages can initiate or promote β-cell auto‐ immunity [75].

**Figure 3.** Information brought by animal models regarding coxsackievirus B infection and some aspects of type 1 dia‐ betes pathogenesis

#### **5.2. In vitro infection of β-cells and other cells with enteroviruses**

Experiments have been conducted in vitro in order to analyse the hypotheses in favour of an association between enterovirus infections and T1D. Whether enteroviruses were able to in‐ fect the pancreatic tissue is a key issue concerning the relationship between enteroviruses and T1D. It has been shown that enteroviruses may be involved in the pathogenesis of T1D, either through direct β-cell infection or as triggers of the autoimmune processes. In particu‐ lar, some results from in vitro experiments have suggested that enteroviruses, and especially CVB, may infect human β-cells and the infection may result in no apparent immediate effect or in functional impairment of β-cell [175, 174, 167, 124]. Most common enteroviruses in the environment can infect cultured human islets with β-cell destruction [93]. The figure 4 sum‐ marizes information brought by in vitro studies regarding coxsackievirus B infection which can be relevant for type 1 diabetes pathogenesis.
