Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients.
Journal: 2007/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic beta cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between beta cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of beta cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect beta cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect beta cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.
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Proc Natl Acad Sci U S A 104(12): 5115-5120

Coxsackie B4 virus infection of β cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

+6 authors
Department of Internal Medicine, Endocrine and Metabolic Sciences, and Biochemistry, University of Siena, 53100 Siena, Italy;
Cellular Microbiology and Bioinformatics Unit, Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy;
Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center, 2333, Leiden, The Netherlands;
Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, 56126 Pisa, Italy;
Department of Experimental Medicine and Pathology, University of Rome “La Sapienza,” 00185 Rome, Italy; and
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada T6G 2E1
To whom correspondence should be addressed. E-mail: moc.sitravon@ilouppar.onir

Contributed by Rino Rappuoli, January 19, 2007

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Author contributions: S.C. and A.G.S.v.H. contributed equally to this work; F.D., M.M., R.R., B.O.R., and P.M. designed research; F.D., S.C., A.G.S.v.H., L.M., S.D., F.M., U.B., A.O.M., S.D.P., J.F.E., A.C., B.O.R., and P.M. performed research; and F.D. and R.R. wrote the paper.

Received 2006 Dec 13

Abstract

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic β cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between β cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of β cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect β cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

Keywords: Coxsackie B4 enterovirus, type 1 diabetes
Abstract

Type 1 diabetes mellitus is believed to result from the selective autoimmune destruction of pancreatic islet β cells, occurring in genetically predisposed subjects, and possibly triggered or accelerated by environmental agents (13). One of the environmental risk factors identified by several independent studies is represented by enteroviral infection (4). Epidemiological data showed an increased incidence of type 1 diabetes after epidemics due to enteroviruses, and enteroviral RNA has been detected in the blood of >50% of type 1 diabetes patients at the time of disease onset (5).

Coxsackie virus B4 has been isolated from patients with acute onset type 1 diabetes (6), and some of these isolates have been reported to cause diabetes in mice (7). In cultured human islet cells, several enterovirus strains can replicate, suppress insulin release, and, in a few cases, cause β cell destruction (8).

Enteroviruses may trigger or accelerate the pathological events leading to clinical type 1 diabetes by several mechanisms, which are not necessarily mutually exclusive (4, 7, 8). First, infected islet β cells could be directly destroyed by virus-induced cytolysis. Second, a less aggressive infection could cause an inflammatory reaction in the islets, leading to subclinical levels of β cell destruction and subsequent release of normally sequestered antigens, which then trigger pathogenic autoreactive T cell responses. Alternatively, cross-reactive T cells could be induced, which occurs when viral antigens and self-antigens share antigenic determinants (9, 10).

To date, a direct link between β cell enterovirus infection in vivo, β cell dysfunction, islet inflammation, and β cell destruction has not been established in humans, although signs of enteroviral infection have been recently reported in a small subset of pancreata obtained at autopsy from type 1 diabetic subjects (11). In search for the relationship between viral infection, insulitis, autoimmunity, and β cell function and survival in type 1 diabetes, we studied islets from six recent-onset type 1 diabetic patients (Table 1) collected over a period of 4 years and from 26 control multiorgan donors. Indeed we found that β cells from three of six diabetic patients showed signs of enteroviral infection associated with natural killer (NK) cell islet infiltration. Virus was isolated from infected islets of a patient, sequenced, and identified as a Coxsackie B4. In addition, isolated virus was able to in vitro infect β cells from nondiabetic multiorgan donors, causing β cell dysfunction characterized by impaired glucose-stimulated insulin release.

Table 1.

Characteristics of type 1 diabetic patients studied

Patient no.Age, yrSexTime from diagnosisβ cell functionEnteroviral infection of β cellsβ cell destructionInsulitis
126F*ImpairedYesNoMinor; dominated by NK cells; no evidence for autoreactive T cells
219M9 monthsPartially lostYesNoMinor; dominated by NK cells; no evidence for autoreactive T cells
315F1 weekLostYesLimitedMinor; dominated by NK cells; no evidence for autoreactive T cells
414F8 monthsLostNoYesModerate T cell infiltrate; no NK cells
55M1 weekLostNoYesModerate T cell infiltrate; no NK cells
64F1 weekLostNoYesModerate T cell infiltrate; no NK cells

M, male; F, female.

*Patient 1 was a recipient of a whole pancreas graft, which was removed after 38 months because of recurrent urinary tract infections.

M, male; F, female.

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Acknowledgments

We thank Dr. A. Scipioni for immunohistochemical studies, G. Duinkerken for the autoreactive T cell studies, Drs. F. Maggi and M. Giorgi for virus culture and in vitro islet infection, and Dr. J. Lakey (Clinical Islet Transplant Center, University of Alberta) for providing paraffin sections of 16 control pancreata for this study. We give special thanks to Dr. Silvia Guidotti and Dr. Alessandro Muzzi of the Cellular Microbiology and Bioinformatic Unit of Novartis Vaccines for their invaluable help in sequence retrieval and phylogenetic analyses. This work was supported by grants from the Juvenile Diabetes Research Foundation International, the European Foundation for the Study of Diabetes (a European Foundation for the Study of Diabetes/Pfizer research grant), the Italian Ministry of Health, the Dutch Diabetes Research Foundation, the Promoter Foundation ONLUS, and the Canadian Institutes of Health Research.

Acknowledgments

Abbreviations

NKnatural killer
PBMCperipheral blood mononuclear cell.
Abbreviations

Footnotes

The authors declare no conflict of interest.

Data deposition: The sequence reported in this paper for the Coxsackie B4 genome (Tuscany isolate) has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ480420","term_id":"94449174","term_text":"DQ480420"}}DQ480420).

This article contains supporting information online at www.pnas.org/cgi/content/full/0700442104/DC1.

Footnotes

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