*5.2.1. Enterovirus infection of β-cells*

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

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‐

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

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

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

can be relevant for type 1 diabetes pathogenesis.

autoimmune process [28, 151].

immunity [75].

50 Type 1 Diabetes

betes pathogenesis

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 (isletreactive) CD4+ TH1 cells [20].

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

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

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 β-cells. In addition, the activation of MHC molecules in human fetal islet cells cultures in‐ fected by CVB4 could result in an increase exposure of infected cells to the immune system and support the autoimmune response against β-cells [119].

induce a virus-specific nonresponsiveness [95]. A global analysis of all these findings sug‐ gests that thymus organ can be infected by coxsackievirus B which can disturb the organ

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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

53

The antibody-dependent enhancement (ADE) of infection is a mechanism observed in vitro with various viruses and which can intervene in pathogenic processes induced by these virus‐ es [145]. The ADE of CVB4 infection has been discovered by our group. It is caused by enhanc‐ ing antibodies devoid of neutralizing activity and has been found in serum /plasma of T1D patients and controls. These antibodies, isolated from plasma by affinity chromatography, in‐ crease the CVB4-induced synthesis of IFN-α by human peripheral blood mononuclear cells (PBMC) in vitro [25]. It has been demonstrated that IFN-α synthesis by PBMC infected with CVB4 prealably incubated with specific antibodies is a result of the infection of monocytes that occurs by a mechanism involving the receptors CAR and those for the Fc portion of IgG mole‐ cule, FcγRII (Fcγ receptor II) and FcγRIII localised at the cell surface membrane [66]. CVB4 can strongly induce the production of IFN-α by PBMCs from patients with T1D compared with PBMC from healthy controls, which is due to anti-CVB4 enhancing antibodies bound to the cell surface membrane. In addition, a higher level of IFN-α was produced by PBMC of patients in‐ oculated with CVB4 prealably incubated with plasma of patients [67]. The target of these anti‐ bodies has been identified as the enteroviral protein VP4 and it has been shown that the prevalence and the titres of anti-VP4 antibodies were higher in patients with T1D than in con‐ trol subjects [26]. Specific anti-VP4 antibodies enhance the infection of PBMC with CVB4 [129]. The sequence of VP4 recognized by these antibodies was investigated and identified in com‐ petition experiments as amino acids 11 to 30 by using synthetic overlapping peptides span‐ ning CVB4E2 VP4 protein [130]. The VP4 protein and a VP4 peptide have been used to detect anti-CVB4 enhancing antibodies by ELISA [26, 130]. The fact that enhancing anti-CVB4 anti‐ bodies bind the viral particles through VP4 is challenging, since, according the structural anal‐ ysis of frozen enteroviruses by X-ray diffraction, the capsid protein VP4 is localized along the inner virion surface, like the amino-terminal sequences of the three external proteins VP1, VP2 and VP3. The explanation lies in the dynamic character of the virus structure at 37°C that would allow an exposure of these normally internal sequences and making a piece of the VP4 protein accessible to antibodies, as it has been shown in the case of the amino-terminal part of

The increased infection of monocytes with CVB4 due to enhancing antibodies could lead, in vivo, in dissemination and worsening of histological lesions that may contribute to CVB4 induced disease, as described in a model of CVB3-induced myocarditis [131, 58, 92]. Further‐ more, the enterovirus –induced production of IFN-α enhanced by antibodies, can play a pathogenic role. Indeed, chronic IFN-α synthesis or its abnormal activation in response to recurrent or repeated enteroviral infections can be associated with disorders leading to auto‐

Further studies are needed to investigate the role of enhancing antibodies in the CVB-in‐

function with possible effects on the autoimmune processes leading to T1D.

*5.2.3. Antibody-dependent enhancement of enterovirus infection*

VP1 in the poliovirus system [96].

immune diseases [23].

duced pathogenesis of T1D.

The inflammation of β-cells is supposed to be an early event in the pathogenesis of type 1diabetes [45]. An exaggerated inflammatory response to enterovirus may contribute to in‐ duce a prolonged inflammation state and β-cell loss, and could initiate or aggravate patho‐ genic processes of type 1 diabetes.

#### *5.2.2. Enterovirus infection of thymus*

It has been shown in mouse that CVB could infect the thymus with a disruption of organ functions that was associated with diabetes [22]. Further studies have been conducted to in‐ vestigate the mechanisms and consequences of infection of thymus with CVB. The establish‐ ment of central T-cell tolerance is ensured by the thymus. Thymic epithelial cells (TEC) participate actively in the development of a biochemical environment needed for the matu‐ ration of immunocompetent T cells. Thymic epithelial cells are actively involved in the pro‐ motion of T-cell maturation by mediating negative and positive selection of thymocytes and by participating to the induction of tolerance [136].

In collaboration with Pr Vincent Geenen and his team (University of Lièges, Belgium) we in‐ vestigated the hypothesis that T1D which is an autoimmune disease, can result from the dis‐ turbance of the central tolerance. Due to the role of thymus in induction and establishment of self-tolerance, enteroviral infection of TEC may result in interference and disturbance of T-cell ontogeny, which can induce or enhance the immune process leading to T1D. The infection of human TEC primary cultures with CVB4 and the resulting consequences on TEC function have been studied. Human TEC, isolated from thymus fragments obtained from children un‐ dergoing corrective surgery, were infected with CVB4 JVB and E2 strains. Findings from this study have revealed that a cytolytic virus such as CVB4 can infect persistently human TEC cul‐ tures without obvious cytopathic effect and this infection have led to a continuous increased production of cytokines IL-6, GM-CSF and LIF [14]. In order to evaluate the effect of enterovi‐ rus infection onto fetal thymus during pregnancy, intact explanted human foetal thymic or‐ gan cultures were infected with CVB4E2 strain. Results from this study have shown progressive thymocyte depletion and upregulation of MHC-I molecules expression on CD4+CD8+ double positive cells [15]. Another study was conducted on mouse to assess the ef‐ fect of CVB infection on thymocytes maturation and differentiation. In this study, whole foetal thymus organ cultures obtained from 14 days foetal CD-1 mice were infected with CVB4E2 strain. Findings from that study have revealed in infected culture a disturbance of maturation and differentiation of T cells characterized by increased levels of mature CD4+ and CD8+ cells associated with decreased percentage of double positive cells [16].

Furthermore it was reported that CVB4 RNA can be found in thymus up to 70 days after per os infection of mice with CVB4E2. In vitro, CVB4 was able to infect and replicate in primary cultures of adult murine splenic and thymic cells [81].

The ability of enteroviruses such as CVB4 to infect the thymus during fetal life could have deleterious effects on thymus functions, since neonatal exposure to thymotropic virus could induce a virus-specific nonresponsiveness [95]. A global analysis of all these findings sug‐ gests that thymus organ can be infected by coxsackievirus B which can disturb the organ function with possible effects on the autoimmune processes leading to T1D.

### *5.2.3. Antibody-dependent enhancement of enterovirus infection*

β-cells. In addition, the activation of MHC molecules in human fetal islet cells cultures in‐ fected by CVB4 could result in an increase exposure of infected cells to the immune system

The inflammation of β-cells is supposed to be an early event in the pathogenesis of type 1diabetes [45]. An exaggerated inflammatory response to enterovirus may contribute to in‐ duce a prolonged inflammation state and β-cell loss, and could initiate or aggravate patho‐

It has been shown in mouse that CVB could infect the thymus with a disruption of organ functions that was associated with diabetes [22]. Further studies have been conducted to in‐ vestigate the mechanisms and consequences of infection of thymus with CVB. The establish‐ ment of central T-cell tolerance is ensured by the thymus. Thymic epithelial cells (TEC) participate actively in the development of a biochemical environment needed for the matu‐ ration of immunocompetent T cells. Thymic epithelial cells are actively involved in the pro‐ motion of T-cell maturation by mediating negative and positive selection of thymocytes and

In collaboration with Pr Vincent Geenen and his team (University of Lièges, Belgium) we in‐ vestigated the hypothesis that T1D which is an autoimmune disease, can result from the dis‐ turbance of the central tolerance. Due to the role of thymus in induction and establishment of self-tolerance, enteroviral infection of TEC may result in interference and disturbance of T-cell ontogeny, which can induce or enhance the immune process leading to T1D. The infection of human TEC primary cultures with CVB4 and the resulting consequences on TEC function have been studied. Human TEC, isolated from thymus fragments obtained from children un‐ dergoing corrective surgery, were infected with CVB4 JVB and E2 strains. Findings from this study have revealed that a cytolytic virus such as CVB4 can infect persistently human TEC cul‐ tures without obvious cytopathic effect and this infection have led to a continuous increased production of cytokines IL-6, GM-CSF and LIF [14]. In order to evaluate the effect of enterovi‐ rus infection onto fetal thymus during pregnancy, intact explanted human foetal thymic or‐ gan cultures were infected with CVB4E2 strain. Results from this study have shown progressive thymocyte depletion and upregulation of MHC-I molecules expression on CD4+CD8+ double positive cells [15]. Another study was conducted on mouse to assess the ef‐ fect of CVB infection on thymocytes maturation and differentiation. In this study, whole foetal thymus organ cultures obtained from 14 days foetal CD-1 mice were infected with CVB4E2 strain. Findings from that study have revealed in infected culture a disturbance of maturation and differentiation of T cells characterized by increased levels of mature CD4+ and CD8+ cells

Furthermore it was reported that CVB4 RNA can be found in thymus up to 70 days after per os infection of mice with CVB4E2. In vitro, CVB4 was able to infect and replicate in primary

The ability of enteroviruses such as CVB4 to infect the thymus during fetal life could have deleterious effects on thymus functions, since neonatal exposure to thymotropic virus could

and support the autoimmune response against β-cells [119].

genic processes of type 1 diabetes.

52 Type 1 Diabetes

*5.2.2. Enterovirus infection of thymus*

by participating to the induction of tolerance [136].

associated with decreased percentage of double positive cells [16].

cultures of adult murine splenic and thymic cells [81].

The antibody-dependent enhancement (ADE) of infection is a mechanism observed in vitro with various viruses and which can intervene in pathogenic processes induced by these virus‐ es [145]. The ADE of CVB4 infection has been discovered by our group. It is caused by enhanc‐ ing antibodies devoid of neutralizing activity and has been found in serum /plasma of T1D patients and controls. These antibodies, isolated from plasma by affinity chromatography, in‐ crease the CVB4-induced synthesis of IFN-α by human peripheral blood mononuclear cells (PBMC) in vitro [25]. It has been demonstrated that IFN-α synthesis by PBMC infected with CVB4 prealably incubated with specific antibodies is a result of the infection of monocytes that occurs by a mechanism involving the receptors CAR and those for the Fc portion of IgG mole‐ cule, FcγRII (Fcγ receptor II) and FcγRIII localised at the cell surface membrane [66]. CVB4 can strongly induce the production of IFN-α by PBMCs from patients with T1D compared with PBMC from healthy controls, which is due to anti-CVB4 enhancing antibodies bound to the cell surface membrane. In addition, a higher level of IFN-α was produced by PBMC of patients in‐ oculated with CVB4 prealably incubated with plasma of patients [67]. The target of these anti‐ bodies has been identified as the enteroviral protein VP4 and it has been shown that the prevalence and the titres of anti-VP4 antibodies were higher in patients with T1D than in con‐ trol subjects [26]. Specific anti-VP4 antibodies enhance the infection of PBMC with CVB4 [129]. The sequence of VP4 recognized by these antibodies was investigated and identified in com‐ petition experiments as amino acids 11 to 30 by using synthetic overlapping peptides span‐ ning CVB4E2 VP4 protein [130]. The VP4 protein and a VP4 peptide have been used to detect anti-CVB4 enhancing antibodies by ELISA [26, 130]. The fact that enhancing anti-CVB4 anti‐ bodies bind the viral particles through VP4 is challenging, since, according the structural anal‐ ysis of frozen enteroviruses by X-ray diffraction, the capsid protein VP4 is localized along the inner virion surface, like the amino-terminal sequences of the three external proteins VP1, VP2 and VP3. The explanation lies in the dynamic character of the virus structure at 37°C that would allow an exposure of these normally internal sequences and making a piece of the VP4 protein accessible to antibodies, as it has been shown in the case of the amino-terminal part of VP1 in the poliovirus system [96].

The increased infection of monocytes with CVB4 due to enhancing antibodies could lead, in vivo, in dissemination and worsening of histological lesions that may contribute to CVB4 induced disease, as described in a model of CVB3-induced myocarditis [131, 58, 92]. Further‐ more, the enterovirus –induced production of IFN-α enhanced by antibodies, can play a pathogenic role. Indeed, chronic IFN-α synthesis or its abnormal activation in response to recurrent or repeated enteroviral infections can be associated with disorders leading to auto‐ immune diseases [23].

Further studies are needed to investigate the role of enhancing antibodies in the CVB-in‐ duced pathogenesis of T1D.

protective effect in mouse model. Therefore, further studies are needed to understand the

The authors thank Delphine Caloone for technical assistance and all their collaborators. The studies performed by the authors or in progress have been or are supported by EU FP5 VIR‐ DIAB Project (Contract QLK 2-CT-2001-01910), EU FP6 Integrated Project EURO-THY‐ MAIDE, (Contract LSHB-CT-2003-503410), EU FP7 PEVNET Project (FP7-HEALTH-2010 single-stage N° 261441), grants from Nord-Pas-de-Calais Région (ArCir convention 2004/018; BBS 2006), CHRU Lille, the ministère de l'Education nationale de la recherche et de la technologie, Université de Lille 2, France, and the comité mixte de coopération univer‐ sitaire franco-tunisien (CMCU 2004 N◦ 04/G0810 and CMCU 2008N808/G0808). Didier Hob‐ er was Fondation pour la Recherche Médicale 2008 prize winner. Didier Hober is a member

mechanisms behind this complex relationship between enteroviruses and T1D.

of the VIrus in Diabetes International Study group (VIDIS group).

, Enagnon Kazali Alidjinou1

\*Address all correspondence to: didier.hober@chru-lille.fr

, Karena Riedweg1

1 Université Lille 2, CHRU, Laboratoire de Virologie/ EA3610, 59037 Lille, France

immunity associated with type 1 diabetes. *Diabetologia* 51(5), 769-72.

2 UPJV CHU, Service d'Endocrinologie-Diabétologie-Nutrition, 80054 Amiens, France

[1] Aarnisalo, J., Veijola, R., Vainionpaa, R., Simell, O., Knip, M., & Ilonen, J. (2008). Cy‐ tomegalovirus infection in early infancy: risk of induction and progression of auto‐

[2] Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D., & Brown, F. (1989). The threedimensional structure of foot-and-mouth disease virus at 2.9 A resolution. *Nature*

, Ilham Moumna1

and Rachel Desailloud2

, Anne Goffard<sup>1</sup>

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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

55

,

**Declaration of interest**

No conflict of interest

**Acknowledgements**

**Author details**

Laura Choteau1

**References**

Didier Hober1\*, Famara Sané1

337(6209), 709-16.

**Figure 4.** Information brought by in vitro studies regarding coxsackievirus B infection

#### **5.3. Complex relationship between enteroviruses and type 1 diabetes**

As mentioned above, the role of enteroviruses in T1D disease is strongly suspected. In con‐ trast, a protective role of enteroviruses is suggested as well. Experimental data in favour of a protective role of these viruses have been reported. Indeed, some studies have shown that, rather than triggering an autoimmune process, CVB infections can provide significant pro‐ tection against the development of T1D [155, 48]. Coxsackievirus B4, the human enterovirus most associated with an etiologic role in human T1D, has been reported to increase the rate of diabetes onset in older NOD mice but not in younger mice [134]. This result has been con‐ firmed by other groups who provided evidence that disease induction required a pre-exist‐ ing accumulation of β-cell specific autoreactive T cells within the pancreas, a phenomenon observed in older NOD mice, but not in younger mice [74, 156]. This protective effect may involve the virus strain, its virulence and replication rate, as well as the stage of autoim‐ mune development, and the mechanism relies in long-term tolerance due to an increase in protective regulatory T cells with TGF-β production [49].

These findings support the concept that virus infections occurring early in childhood had a protective effect against T1D and are in agreement with the hygiene hypothesis [7, 157]. In‐ deed, it should be emphasized that there are significantly more enterovirus infections annu‐ ally than new cases of T1D in population. The decreased enterovirus exposure rates following the increased hygiene levels might explain the high risk of developing the disease, since it has been revealed in epidemiological studies that T1D incidence is higher in devel‐ oped countries than in developing ones, from less than 1 per 100,000 inhabitants in Asia to 14 in US and even more than 30 per 100,000 in Scandinavia [139].
