receptor sirp polymorphism in the innate immune

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    MouseAutoimmunity in the Nonobese Diabetic

    Controls CD47 Binding andReceptor SIRPPolymorphism in the Innate Immune

    S. DanskaJayneAngelo J. Canty, Omid Gulban, David R. Greaves and

    Andrea Sut Ling Wong, Steven Mortin-Toth, Michael Sung,


    published online 10 October 2014J Immunol




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  • The Journal of Immunology

    Polymorphism in the Innate Immune Receptor SIRPaControls CD47 Binding and Autoimmunity in the NonobeseDiabetic Mouse

    Andrea Sut Ling Wong,*, Steven Mortin-Toth, Michael Sung, Angelo J. Canty,

    Omid Gulban, David R. Greaves,x and Jayne S. Danska*,,{

    The signal regulatory protein (SIRP) locus encodes a family of paired receptors that mediate both activating and inhibitory signals

    and is associated with type 1 diabetes (T1D) risk. The NOD mouse model recapitulates multiple features of human T1D and

    enables mechanistic analysis of the impact of genetic variations on disease. In this study, we identify Sirpa encoding an inhibitory

    receptor on myeloid cells as a gene in the insulin-dependent diabetes locus 13.2 (Idd13.2) that drives islet inflammation and T1D.

    Compared to T1D-resistant strains, the NOD variant of SIRPa displayed greater binding to its ligand CD47, as well as enhanced

    T cell proliferation and diabetogenic potency. Myeloid cellrestricted expression of a Sirpa transgene accelerated disease in a dose-

    dependent manner and displayed genetic and functional interaction with the Idd5 locus to potentiate insulitis progression. Our

    study demonstrates that variations in both SIRPa sequence and expression level modulate T1D immunopathogenesis. Thus, we

    identify Sirpa as a T1D risk gene and provide insight into the complex mechanisms by which disease-associated variants act in

    concert to drive defined stages in disease progression. The Journal of Immunology, 2014, 193: 000000.

    Type I diabetes (T1D) is caused by autoreactive T cells thatinfiltrate and destroy pancreatic b cells, resulting in lossof insulin production and dependence on exogenous in-

    sulin. The NOD mouse develops spontaneous autoimmune T1Dthat shares many features with the human disease (1). In humansand NOD mice, T1D is a complex disease, resulting from inter-action between multiple susceptibility genes and poorly definedenvironmental modifiers. The greatest genetic risk factor for T1Din humans and NOD mice is MHC class II (MHC II) haplotypes,in which the two species share remarkably similar amino acidresidues that influence peptide binding and Ag presentation toT cells. In addition to the MHC II, known causal variants includeregulator of immune homeostasis IL-2 and IL-2R and inhibitors ofT cell activation CTLA-4 and LYP (2). These disease-causingvariants are common functional alleles involved in maintaining

    the balance between tolerance and immunity, suggesting that T1Ddevelops due to dysregulation of the immune response.Genome-wide association studies (GWAS) in humans have

    identified .40 single nucleotide polymorphisms (SNPs) associ-ated with T1D susceptibility, including polymorphisms in thesignal regulatory protein (SIRP) locus that encodes a family ofactivating and inhibitory receptors (3). Members of the pairedreceptor family have similar extracellular regions but differentsignaling potentials that may have evolved to maintain the balancebetween tolerance and immunity in response to evolutionarypressure such as pathogens (4, 5). The majority of T1D-associatedhuman SNPs have small effect sizes in which each confers modestimpact on the phenotype: to date, few causal genetic variationshave been identified. Collectively, GWAS fail to fully explain theheritability of T1D likely because this approach did not capturethe contribution of genegene and geneenvironment interactionsunderlying the complex disease phenotype (6). As shown in thisstudy, the NOD mouse model enables analysis of complex featuresof T1D heritability through enriched preclinical phenotypes, ac-cess to immune cell compartments, and germline manipulations totest the effects of both individual and combined genetic variationson disease.Diabetes onset in NOD mice is preceded by islet inflammation

    (insulitis) by multiple leukocyte subsets. Insulitis begins withperi-islet accumulation of myeloid cells and T cells, which theninvades the b-cellrich interior (invasive insulitis), leading toeventual loss of insulin secretion. In contrast, the nonobese T1D-resistant (NOR) strain displays only peri-islet infiltration by APCthat does not invade the islet interior or compromise b cellfunction (7). The NOR strain is 88% identical by descent toNOD, shares the high risk Mhc haplotype, but is protected fromT1D by genomic inheritance from C57BLKS/J mice (8). Previ-ously, we showed that progression from peri-islet to invasiveinsulitis in NOD mice was regulated by two Idd loci, Idd5 andIdd13 (7). In this study, we report high-resolution genomic,genetic, and functional analyses supporting Sirpa, encoding thesignal inhibitory receptor protein (SIRPa) as the candidate gene

    *Department of Immunology, Faculty of Medicine, University of Toronto, Toronto,Ontario M5S1A8, Canada; Program in Genetics and Genome Biology, TheHospital for Sick Children Research Institute, Toronto, Ontario M5G1X8, Can-ada; Department of Mathematics and Statistics, McMaster University, Hamilton,Ontario L8S4L8, Canada; xSir William Dunn School of Pathology, University ofOxford, Oxford OX13RE, United Kingdom; and {Department of Medical Biophys-ics, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S1A8, Canada

    Received for publication August 12, 2014. Accepted for publication September 8,2014.

    This work was supported by grants from the Juvenile Diabetes Research Foundation,Genome Canada (administered through the Ontario Genomics Institute), and theCanadian Institutes of Health Research (to J.S.D.).

    Address correspondence and reprint requests to Dr. Jayne S. Danska, The Hospital forSick Children, Peter Gilgan Research Tower, 686 Bay Street, 15th Floor, Toronto, ONM5G 0A4, Canada. E-mail address:

    The online version of this article contains supplemental material.

    Abbreviations used in this article: T1D, type 1 diabetes; DC, dendritic cell; EAE,experimental autoimmune encephalomyelitis; GWAS, genome-wide associationstudies; hCD47-Fc, human CD47-Fc protein; IgV, Ig variable; LN, lymph node;b2m, b2-microglobulin; Mf, macrophage; mCD47-Fc, mouse CD47-Ig variabledomainhuman IgG-Fc fusion protein; MHC II, MHC class II; NOR, nonobese type 1diabetesresistant; PDCA-1, plasmacytoid DC Ag-1; PLN, pancreatic LN; SIRP, signalregulatory protein; SNP, single nucleotide polymorphism.

    Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

    Published October 10, 2014, doi:10.4049/jimmunol.1401984 by guest on A

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  • at Idd13.2, which interacts with the Idd5 locus to drive insulitisprogression and T1D.

    Materials and MethodsMice

    All mice used in this study were maintained in specified pathogen-freeconditions at The Hospital for Sick Children (Toronto, Ontario, Canada)and followed the guidelines for the institutional animal care committee.

    Generation of Idd13 congenic mice

    All NOD.NOR-Idd13 congenic mice were generated by microsatellitemarker-directed selection of breeders (Supplemental Table I). NOR.NOD-Idd5, NOR.NOD-Idd13, and NOR.NOD-Idd5.Idd13 congenic mice weregenerated using similar strategies (Supplemental Table II). Microsatellitemarkers were developed using diNucleotide


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