Type 1 DM Is Associated With Enterovirus in Gut Mucosa

In This Article

Abstract and Introduction

Abstract

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months' observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Introduction

Type 1 diabetes is one of the most common chronic diseases in developed countries, and its incidence has increased sharply since the second world war. It is caused by a selective destruction of insulin-producing β cells in the pancreas mediated by immunological mechanisms. Susceptibility to the disease is modulated by >40 different risk genes, with HLA genes contributing more than half of the genetic susceptibility. Environmental factors clearly influence the disease risk and contribute to the rapidly increasing incidence rates.

The connection between type 1 diabetes and enterovirus infections has been widely studied. Enteroviruses have been found in the blood and pancreas of type 1 diabetic patients in several studies, and they have also been associated with increased risk of type 1 diabetes in prospective studies.^[1–7] The recent discovery of the genetic polymorphisms of IFIH1 gene as diabetes risk-modulating factors has further strengthened this association, since these genes mediate the recognition of enteroviruses by the innate immune system.^[8,9] Diabetes-associated polymorphisms seem to be associated with a strong innate immune response, which may lead to enhanced inflammation response during virus infection.^[10] Other innate immune system genes (IRF7 network) also modulate the risk of T1D.^[11]

Frequent detection of the virus in diabetic patients has suggested a possible role of viral persistence. Enterovirus persistence has previously been described, e.g., in chronic cardiomyopathies^[12] and in animal models,^[13–16] where enterovirus and other picornaviruses may persist for prolonged periods. These studies have suggested that enterovirus can persist as double-stranded RNA without clear protein synthesis.^[15,17] It is known from several viral diseases that persistent infection typically leads to a strong innate immune response, local inflammation, and immune-mediated tissue damage.

Celiac disease is characterized by small intestinal inflammation, mucosal damage, and increased mucosal permeability. The association between type 1 diabetes and celiac disease is well established. It is therefore intriguing to hypothesize whether enterovirus infection and enteropathy contribute to this association.

In spite of the recent progress in this research field, it is not known how enteroviruses could cause selective damage in insulin-producing β cells. Their primary replication site is the gut mucosa, from which the virus can spread to the blood (primary viremia) and infect internal organs. However, the possible presence of enteroviruses in human intestine has not been studied in detail. We recently published the first such study suggesting that enterovirus may be present in the small intestine in type 1 diabetic patients.^[18] The current study evaluates the hypothesis that gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients, maintaining a continuous inflammatory state that can spread to the pancreas and play a crucial role in enterovirus-induced diabetes.

Table 1. Summary of study subjects
	T1D patients*	Celiac disease patients	Control subjects
Subjects (N)	39	40	41
Males/females (N/N)	11/28	13/27	16/25
Age (years)	43 (18–63)	41 (18–75)	47 (23–76)
HLA DQ2 and/or DQ8 (%)	57	100	34
Age at diabetes diagnosis (years)**	18 (4–51)
Diabetes duration (years)**	20 (0–38)

Data are median (range) unless otherwise indicated. T1D, type 1 diabetic. *Sixteen of the type 1 diabetic patients also had celiac disease. **Exact information from only 23 type 1 diabetic patients available. Others were diagnosed early in childhood.

Table 2. Subjects positive for enterovirus RNA by ISH in small intestine biopsy samples
	All positive	Weak positive	Moderate positive	Strong positive
T1D patients (N = 39)	29 (74)**	9 (23)	16 (41)**	4 (10)*
Celiac disease patients (N = 40)	18 (45)	13 (33)	3 (8)	2 (5)
Control subjects (N = 41)	12 (29)	10 (24)	2 (5)	0 (0)

Data are N (%). Weak positive, an average of 1–10 positive cells per field; moderate positive, 10–100 positive cells per field; strong positive, >100 cells per field. T1D, type 1 diabetic. **P < 0.001 compared with control subjects. *P = 0.035 compared with control subjects.

Table 3. Inflammation markers in ISH+ and ISH2 subjects
	ISH+	ISH2	P
N	47	57
CD3 IELs	64	49	0.16
αβ IELs	64	51	0.23
γδd IELs	66	42	0.02
γδ/CD3	0.24	0.17	0.04
HLA-DR	62	37	0.02
IgA deposits	54	31	0.02

Data are percent positive unless otherwise indicated.

Table 4. Subjects with increased density of CD3, ab and gd T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits in the gut mucos ^a
	CD3	αβ	γδ	HLA-DR	IgA deposits
All T1D patients (N = 23)	35	30	52***	48**	42***
T1D patients without celiac disease (N = 17)	18	12	35	29	10*
Celiac disease patients (N = 40)	88***	83***	93***	79***	100***
Control subjects (N = 41)	37	46	15	18	0

Data are percent. T-cell markers were analyzed from 23 type 1 diabetic patients and all other study subjects. HLA-DR expression was analyzed from 23 type 1 diabetic patients, 38 celiac disease patients, and 38 control subjects, and IgA deposits were analyzed from 24 type 1 diabetic patients, 30 celiac disease patients, and 41 control subjects. The following cut-off values were used for positivity: CD3 cells (>37 cells/mm), ab-positive cells (>25 cells/mm), and gd-positive cells (>4.3 cells/mm). T1D, type 1 diabetic. ***P < 0.001 compared with control subjects. **P = 0.015 compared with control subjects. *P < 0.05 compared with control subjects.

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