Type 1 DM Is Associated With Enterovirus in Gut Mucosa
The current study suggests that the intestinal mucosa of type 1 diabetic patients is frequently infected by enteroviruses. The findings fit well with viral persistence, a phenomenon commonly observed in picornavirus infections and that can lead to prolonged inflammation and tissue damage. This observation also confirms our earlier study showing a similar phenomenon in a smaller subset of diabetic patients and control subjects.
Several factors suggest that this observation may have clinical significance. First, the presence of enterovirus RNA was associated with increased inflammation activity in the gut mucosa, including both cell-mediated and antibody-mediated inflammation (accumulation of IELs and IgA deposits). Second, the virus was more frequently detected in type 1 diabetic patients than in the control subjects, suggesting that it may play a role in the disease process. Third, our findings are consistent with prolonged infection and viral persistence in gut mucosa: the virus was found in the follow-up sample taken after 1 year's observation in all three diabetic patients who were initially virus positive and from whom such follow-up samples were available. Viral persistence was also supported by the fact that the viral genome (RNA) occurred frequently in the absence of viral proteins, and this was seen more often in the patient group than in the control group. Such an imbalance between viral RNA and protein expression has earlier been linked to enterovirus persistence, showing that enterovirus capsid protein synthesis is decreased in persistent infection and that virus replication occurs mainly on the RNA level without producing infective virus. An analogous finding in human endomyocardial tissue shows a presence of enteroviral RNA without production of viral VP1 protein in idiopathic dilated cardiomyopathy and chronic coronary disease. Altogether, the current study fits well with previous observations in orally infected mice showing that enterovirus can persist in intestinal mucosa. Accordingly, the virus detected in these patients may have persisted for a longer period. However, in light of the current study, it is not possible to conclude whether it infected these individuals before or after the diagnosis of type 1 diabetes. Studies have shown that a large proportion of type 1 diabetic patients are already positive for enterovirus in blood at the diagnosis of diabetes, and even earlier during the pathogenesis, close to the appearance of islet autoantibodies.
It is possible that individuals susceptible to type 1 diabetes are prone to enterovirus infections and their persistence. In theory, such susceptibility could be mediated by genetic factors conferring susceptibility to type 1 diabetes. It is known that some of these susceptibility genes, such as HLA and IFIH1 genes, influence immune responses against enteroviruses. However, there are no studies available on the possible role of these genes in the development of enterovirus persistence. In the current study, the detection of enterovirus RNA was not associated with HLA risk genes for type 1 diabetes, arguing against a major effect of HLA genes. In addition to genetic factors, hyperglycemia is known to predispose to infections. Therefore, we cannot exclude the possibility that diabetes itself could predispose to prolonged enterovirus infections. However, the detection of enterovirus was not associated with the duration of type 1 diabetes, which indirectly speaks against this possibility.
The intensity of staining and the number of positive cells in ISH varied between the study groups, with patients with type 1 diabetes showing significantly stronger positivity than celiac disease patients and control subjects. This suggests that the virus replicates at a higher level in diabetic subjects, producing more RNA molecules and infecting more cells. The very weak positives were relatively frequent also among disease control subjects, suggesting that a low-grade infection may have contributed to the nonspecific gastrointestinal symptoms in some of them.
Virus RNA was found using different techniques including ISH and RT-PCR assays. Even though they gave parallel results, it was evident that enterovirus positivity was more frequent in ISH than in RT-PCR. This could be explained by the fact that two separate biopsy samples were needed for these two methods, as well as by the methodological differences in the pretreatment of the samples and possible presence of PCR inhibitors, which impair the sensitivity of PCR.
In conclusion, we propose that persistent enterovirus infection in gut mucosa may play a role in the pathogenesis of type 1 diabetes. Gut mucosa may be an important virus reservoir from which the virus can spread to the pancreas, which is anatomically very close and has common lymphatic and vasculature networks. Also, the viral persistence in gut mucosa may maintain chronic inflammation milieu in this network, which can promote islet autoreactivity by bystander activation mechanism. For example, γδ T cells, which were clearly associated with the presence of enterovirus in intestinal mucosa, facilitate autoimmunity in enterovirus-induced myocarditis through inhibiting T regulatory cell activity.
Discussion
The current study suggests that the intestinal mucosa of type 1 diabetic patients is frequently infected by enteroviruses. The findings fit well with viral persistence, a phenomenon commonly observed in picornavirus infections and that can lead to prolonged inflammation and tissue damage. This observation also confirms our earlier study showing a similar phenomenon in a smaller subset of diabetic patients and control subjects.
Several factors suggest that this observation may have clinical significance. First, the presence of enterovirus RNA was associated with increased inflammation activity in the gut mucosa, including both cell-mediated and antibody-mediated inflammation (accumulation of IELs and IgA deposits). Second, the virus was more frequently detected in type 1 diabetic patients than in the control subjects, suggesting that it may play a role in the disease process. Third, our findings are consistent with prolonged infection and viral persistence in gut mucosa: the virus was found in the follow-up sample taken after 1 year's observation in all three diabetic patients who were initially virus positive and from whom such follow-up samples were available. Viral persistence was also supported by the fact that the viral genome (RNA) occurred frequently in the absence of viral proteins, and this was seen more often in the patient group than in the control group. Such an imbalance between viral RNA and protein expression has earlier been linked to enterovirus persistence, showing that enterovirus capsid protein synthesis is decreased in persistent infection and that virus replication occurs mainly on the RNA level without producing infective virus. An analogous finding in human endomyocardial tissue shows a presence of enteroviral RNA without production of viral VP1 protein in idiopathic dilated cardiomyopathy and chronic coronary disease. Altogether, the current study fits well with previous observations in orally infected mice showing that enterovirus can persist in intestinal mucosa. Accordingly, the virus detected in these patients may have persisted for a longer period. However, in light of the current study, it is not possible to conclude whether it infected these individuals before or after the diagnosis of type 1 diabetes. Studies have shown that a large proportion of type 1 diabetic patients are already positive for enterovirus in blood at the diagnosis of diabetes, and even earlier during the pathogenesis, close to the appearance of islet autoantibodies.
It is possible that individuals susceptible to type 1 diabetes are prone to enterovirus infections and their persistence. In theory, such susceptibility could be mediated by genetic factors conferring susceptibility to type 1 diabetes. It is known that some of these susceptibility genes, such as HLA and IFIH1 genes, influence immune responses against enteroviruses. However, there are no studies available on the possible role of these genes in the development of enterovirus persistence. In the current study, the detection of enterovirus RNA was not associated with HLA risk genes for type 1 diabetes, arguing against a major effect of HLA genes. In addition to genetic factors, hyperglycemia is known to predispose to infections. Therefore, we cannot exclude the possibility that diabetes itself could predispose to prolonged enterovirus infections. However, the detection of enterovirus was not associated with the duration of type 1 diabetes, which indirectly speaks against this possibility.
The intensity of staining and the number of positive cells in ISH varied between the study groups, with patients with type 1 diabetes showing significantly stronger positivity than celiac disease patients and control subjects. This suggests that the virus replicates at a higher level in diabetic subjects, producing more RNA molecules and infecting more cells. The very weak positives were relatively frequent also among disease control subjects, suggesting that a low-grade infection may have contributed to the nonspecific gastrointestinal symptoms in some of them.
Virus RNA was found using different techniques including ISH and RT-PCR assays. Even though they gave parallel results, it was evident that enterovirus positivity was more frequent in ISH than in RT-PCR. This could be explained by the fact that two separate biopsy samples were needed for these two methods, as well as by the methodological differences in the pretreatment of the samples and possible presence of PCR inhibitors, which impair the sensitivity of PCR.
In conclusion, we propose that persistent enterovirus infection in gut mucosa may play a role in the pathogenesis of type 1 diabetes. Gut mucosa may be an important virus reservoir from which the virus can spread to the pancreas, which is anatomically very close and has common lymphatic and vasculature networks. Also, the viral persistence in gut mucosa may maintain chronic inflammation milieu in this network, which can promote islet autoreactivity by bystander activation mechanism. For example, γδ T cells, which were clearly associated with the presence of enterovirus in intestinal mucosa, facilitate autoimmunity in enterovirus-induced myocarditis through inhibiting T regulatory cell activity.