Congenic mouse strains provide a unique resource for genetic dissection and biological characterization of chromosomal regions associated with diabetes progression in the nonobese diabetic (NOD) mouse. Idd11, a mouse diabetes susceptibility locus, was previously localized to a region on chromosome 4. Comparison of a panel of subcongenic NOD mouse strains with different intervals derived from the nondiabetic C57BL/6 (B6) strain now maps Idd11 to an ∼8-Mb interval. B6-derived intervals protected congenic NOD mice from diabetes onset, even though lymphocytic infiltration of pancreatic islets was similar to that found in NOD mice. In addition, neither thymic structural irregularities nor NKT cell deficiencies were ameliorated in diabetes-resistant congenic NOD mice, indicating that Idd11 does not contribute to these abnormalities, which do not need to be corrected to prevent disease.
Due to the inherent difficulties of characterizing type 1 diabetes in humans, many of the insights into type 1 diabetes pathogenesis have come from biological studies of the nonobese diabetic (NOD) mouse. Similar to human diabetes progression, hyperglycemia develops in the NOD mouse due to an autoimmune response that selectively targets insulin-producing β-cells for destruction (rev. in 2,3). Given that other inbred mouse strains do not spontaneously develop diabetes, there must be genetic defects in the NOD mouse that allow β-cell–specific lymphocytes to infiltrate the islet and mediate destruction. Islet-antigen specificity and diabetes susceptibility are attributable to expression of the unique NOD major histocompatibility complex class II allele, I-Ag7, which is associated with an unusual deletion within the NOD I-Eα chain promoter. However, these particular major histocompatibility complex alleles are also found in nonautoimmune prone strains, demonstrating that this locus alone is not sufficient for disease onset (rev. in 2,3). It is now apparent that there are at least 21 susceptibility loci, termed Idd loci, that contribute to diabetes predisposition in the NOD mouse (Idd1–19, rev. in 2; Idd20, rev. in 4; and Idd21, rev. in 5). Congenic NOD mouse strains, harboring chromosomal intervals derived from nondiabetes-prone strains, have confirmed Idd loci on chromosome (Chr) 1, 2, 3, 4, 6, 11, 13, 17, and 18 (Chr1, 2, 3, 4, 6, 17, rev. in 3; Chr11, rev. in 6; Chr13, rev. in 7; and Chr18, rev. in 5).
One of these loci, Idd11, was linked to a region on Chr4 in outcrosses between NOD and the C57BL/6 (B6) and SJL mouse strains (8). As B6 mice were shown to harbor a resistance allele for Idd11, we initially generated three NOD congenic mouse strains that contained different B6-derived intervals for Chr4 on the NOD genetic background. These congenic strains confirmed and localized Idd11 to Chr4 between, but not including, D4Mit119 and D4Mit204 (9). We have recently backcrossed one of these congenic strains (NOD.B6Idd11A) to the NOD strain, and this led to the identification of a pup with a recombination event within the Idd11 interval. This pup was used to establish a new congenic strain (NOD.B6Idd11D) with a smaller B6-derived Chr4 interval compared with the NOD.B6Idd11A interval (Table 1) (Note: While establishing this strain, we identified a discrepancy in the genetic positioning of D4Mit339 and D4Mit69. According to the mouse genome sequence database and our congenic strains, D4Mit339 is proximal to D4Mit69. This marker order agrees with a previous genetic map reported by the MIT genetic database [available at http://www.broad.mit.edu], but disagrees with the current MGI database [Table 1].) NOD.B6Idd11D females were monitored for spontaneous diabetes and demonstrated a significant decrease (from 71 to 17%, P < 0.001) in the incidence of diabetes compared with age-matched NOD females (Fig. 1). This incidence was similar to two previous congenic strains, NOD.B6Idd11A and NOD.B6Idd11C (9). Given that the three protected congenic mouse strains have in common a ∼8-Mb B6-derived interval, we predict that Idd11 is located within this interval (Table 1).
A number of groups have utilized congenic mouse strains to confirm and localize diabetes susceptibility loci (rev. in 3). Idd11 was originally identified in an outcross between NOD and B6 strains and mapped to a region on Chr4 not previously detected in other NOD outcrosses (8). Another outcross, between NOD and C57BL/10 (B10), also demonstrated linkage of type 1 diabetes to Chr4 but not to the Idd11 region. This locus, Idd9, mapped next to the telomere of Chr4 (10). It was not unexpected that B6 and B10, two closely related strains, might yield different linkage results because these two strains were shown to differ at multiple loci on Chr4 (11). Idd11 was subsequently confirmed by a NOD congenic strain harboring a B6-derived interval (∼33 Mb) encompassing the predicted Idd11 locus but excluding the originally described Idd9 locus (9). Our panel of subcongenic strains has now localized Idd11 to a ∼8-Mb interval and suggests that the Idd11 locus may yet represent the effect of one gene because, unlike other Idd loci, the Idd11 effect has not been dissected into separate contributing intervals (rev. in 2,3). In contrast, diabetes-resistant congenic NOD strains, harboring B10-derived intervals, have expanded the Idd9 locus from the telomere to encompass three loci (Idd9.1, Idd9.2, and Idd9.3); one of which, Idd9.1, now is in the same region as the original B6-defined Idd11 interval. The separate Idd9.1 and Idd9.2 effects have not yet been independently confirmed but were inferred by comparing the degree of diabetes resistance between subcongenic strains with different B10-derived Chr4 intervals (12). It remains to be determined if the Idd9.1 and Idd11 effects are due to the same gene. In either case, there are at least 75 genes within the currently defined Idd11 interval, including Lck, an attractive candidate that encodes a protein tyrosine kinase implicated in NOD immunopathology (13). Additional NOD subcongenic strains with smaller B6-derived subintervals will be required to further localize Idd11 and either eliminate or confirm candidate genes for this locus.
Given the number of genes within the defined Idd11 region, biological studies of diabetes-resistant congenic NOD strains may provide important clues for gene identification and underlying cellular mechanisms. Different abnormalities observed in the NOD mouse may affect the lymphocyte repertoire and lead to autoimmunity (3,14). In particular, thymic structural irregularities have been proposed to contribute to dysregulation of NOD central tolerance. The severity of these irregularities has been correlated with age and diabetes progression (15,16). As NOD mice age, the presence of medullary epithelium in the cortex and the development of giant perivascular spaces may affect thymocyte selection and maturation, leading to the escape of autoreactive T-cells. Comparison between immunostained NOD and B6 thymi confirmed the previously reported abnormal thymic architecture. In NOD thymi, the network of thymic epithelium, stained by anti-cytokeratin, is interrupted by enlarged, intralobular, fibroblast-filled, perivascular spaces in the medulla and at the corticomedullary junction (Fig. 2A). Clusters of B-cells, stained by 1D3, are also present and appear to be associated with perivascular space formation (Fig. 2A). The corticomedullary junction is disorganized in the NOD thymi, and ectopic medullary epithelium, defined by MTS-10 staining, can be found scattered within the cortex (Fig. 2B). A novel observation was the staining characteristic of MTS24, a monoclonal antibody that detects thymic epithelial precursors in the fetus (17). Some of these rare cells appeared larger and more brightly stained in NOD thymi compared with B6 thymi (Fig. 2C).
It was hypothesized that correction of NOD thymic structural irregularities may underlie diabetes protection observed in the diabetes-resistant Idd11 congenic mice. NOD.B6Idd11D mice were chosen for further characterization because this strain maintained the smallest B6-derived interval providing the greatest diabetes protection. All thymi taken from NOD.B6Idd11D mice displayed the same degree of abnormal architecture as NOD thymi, including giant perivascular spaces, B-cell clusters, disruption of the corticomedullary junction, and MTS24+ cell staining (Fig. 2). There was also no apparent difference between thymi taken from B6, NOD, and congenic mice for immunostaining of Aire, a transcription factor that regulates the presentation of islet-specific antigens within the thymic stroma (Fig. 2D, ). Heino et al. (19) previously noted abnormal morphology of Aire-positive cells in NOD mice, but this observation was made using a polyclonal rabbit antiserum to Aire, whereas our immunostaining was performed with an anti-Aire rat monoclonal antibody (18). Regardless, it was not possible to differentiate between individual thymic sections from congenic and NOD mice. Flow cytometry also demonstrated that thymic stromal cell populations were not significantly different between congenic and NOD mice (online appendix Table 1 [available at http://diabetes.diabetesjournals.org]). Thus, although 83% of NOD.B6Idd11D mice were protected from diabetes (Fig. 1), all congenic mice examined demonstrated indistinguishable thymic architecture and stromal cell content compared with NOD thymi. It may be that NOD thymic architecture is unrelated to, or a consequence of, disease progression. The rate and composition of thymic export appears normal in pre-diabetic NOD mice (20), and we observed no apparent difference for the number of Aire-expressing cells between thymi taken from NOD and B6 mice. Furthermore, diabetes-resistant congenic NOD mice described by others also displayed thymic disorganization indistinguishable from NOD mice, implying that gross thymic abnormalities do not necessarily need to be corrected to prevent diabetes onset (15).
In addition to thymic abnormalities, Idd11 congenic mice also exhibited extensive insulitis similar to NOD mice. Mild insulitis can be detected in NOD females by 4–5 weeks of age, and complete insulitis is present by 30 weeks of age. Hematoxylin and eosin staining was used to examine pancreata taken from NOD and NOD.B6Idd11D females at 100 and 150 days of age. All pancreata examined demonstrated lymphocytic infiltration of islets (online appendix Fig. 1). Although there was a decrease in the percentage of NOD.B6Idd11D mice at 100 days, exhibiting extensive insulitis compared with NOD mice (28 versus 57%), it was not possible to differentiate between individual NOD.B6Idd11D and NOD pancreata (data not shown). Furthermore, >70% of NOD.B6Idd11D mice exhibited extensive insulitis at 150 days indicating that diabetes protection can occur despite progression of leukocytic infiltration (online appendix Fig. 1). Lyons et al. (12) showed that a B10-derived interval (∼90 Mb) encompassing Idd9.1, Idd9.2, and Idd9.3 was also unable to prevent insulitis but changed the pathogenic profile of the leukocytic infiltrate and prevented diabetes. Likewise, a smaller B6-derived interval (∼11 Mb) for Idd11 appears to enhance the NOD ability to regulate the pathogenic effects of self-reactive lymphocytes that accumulate in the pancreatic islets.
Development of insulitis in Idd11 diabetes-resistant congenic mice indicates that autoreactive T-cells escape from the thymus but are limited in their destructive capability. Regulation of diabetes onset has been correlated with the frequency and stimulation of NKT cells (a CD1d-dependent T-cell subset expressing an invariant Vα14Jα18 TCR α-chain), such that increasing the numbers of NKT cells in ordinarily deficient NOD mice significantly decreases the incidence of diabetes (rev. in 21). The regulation of NKT cell frequency appeared to be controlled by a locus reported to map to a region on Chr4 encompassing Idd11 (22). Therefore, the frequency of these cells within the thymus, spleen, and liver was examined from female NOD.B6Idd11D mice. Unlike B6 mice, congenic mice did not have an increase in the proportion of NKT cells in the thymus, spleen, and liver compared with NOD mice (Fig. 3). If anything, congenic mice appeared to have an even lower proportion of NKT cells, particularly in the thymus and spleen. Although the proportion of NKT cells in liver was not different between congenic and normal NOD mice, the total number of liver lymphocytes was increased in congenic mice. Thus, there was an approximately twofold increase in number of all liver lymphocyte subsets, including T-, B-, and NKT cells (Figs. 3 and data not shown). Despite this nonspecific increase in the number of liver NKT cells, the average number of these cells in congenic mice remained significantly lower than that observed for B6 mice (Fig. 3). Therefore, the NKT cell abnormality was not corrected within these congenic mice.
Recently, Matsuki et al. (22) found that a diabetes-resistant congenic strain, harboring a large B10-derived interval encompassing Idd9 and Idd11, exhibited an increase in percentage and number of CD1d/αGalCer tetramer–positive NKT cells compared with NOD mice. In contrast, we found no change in the percentage of NKT cells in any tissue and no change in the number of these cells in thymus and spleen (Fig. 3). Only an increase in the number of liver NKT cells was observed, reflecting an increase in total liver lymphocytes in our congenic strain and suggesting that the defined Idd11 interval does not control NKT cell frequency. While these opposing results may be due to genetic differences between the derived congenic intervals (i.e., interval size, B10 versus B6 alleles), the overall evidence for genes within Chr4 controlling NKT cell number is not well corroborated. For example, linkage to Chr4 was not found in a (B6xNOD.Nkrp1) × NOD.Nkrp1 backcross in which thymocytes were screened with the CD1d/αGalCer tetramer; nor was there a change in CD4− CD8−/TCRαβ+ thymocytes isolated from a congenic strain similar to the B10-derived Chr4 strain used by Matsuki et al. (22,23,24). Nonobese resistant (NOR) mice, which harbor C57BLKS/J alleles within the defined Idd9 and Idd11 intervals, also exhibited a similar NKT cell deficiency compared with NOD mice (22).
The study presented here has localized Idd11 to an ∼8-Mb interval and demonstrated that neither thymic structural irregularities nor NKT cell deficiencies nor yet insulitis are corrected in Idd11 diabetes-resistant congenic mice. Although these abnormalities may play a role in disease progression, they represent only a subset of the ones that could be investigated (3,14). A different mechanism, possibly involving another described NOD abnormality, exists for protection in the congenic strain, and it is controlled by genetic variation at the Idd11 locus. Consequently, the product of the Idd11 locus can significantly reduce diabetes onset, even in the presence of ongoing autoimmunity (as evidenced by islet infiltrating lymphocytes) and particular immunological abnormalities. Further studies are required to identify the Idd11 effect upon diabetes pathogenesis.
RESEARCH DESIGN AND METHODS
Mice, genotyping, diabetes, and pancreatic histology.
NOD/Lt (NOD) and C57BL/6 (B6) mouse strains were obtained from The Walter and Eliza Hall Institute specific pathogen–free facilities. NOD females in our specific pathogen–free facilities have a cumulative diabetes incidence of 70–80% by 300 days of age; B6 females do not develop diabetes. To generate NOD congenic strains described in this study, (NOD × B6)F1 females were backcrossed to NOD males. Nine subsequent backcrosses were then performed using NOD males or females and appropriate backcross progeny based on genotyping results. B6-derived Chr4 intervals were fixed to homozygosity on the NOD background by brother-sister matings using the tenth backcross generation. NOD.B6Idd11D was generated due to a recombination event within a backcross litter generated from (NOD.B6Idd11A × NOD) × NOD. After an additional backcross to NOD, this particular interval was fixed to homozygosity by brother-sister mating mice that harbored the desired B6-derived interval. NOD.B6Idd11A is a previously described congenic strain (9). Mice were genotyped and monitored for spontaneous diabetes as previously described (7,9). Pairwise comparisons of diabetes incidence for congenic and NOD mouse strains were done using the log-rank test. Pancreata were prepared using Bouin’s solution and were paraffin embedded. Tissue sections (5 μm) were stained with hematoxylin and eosin. At least three noncontiguous sections of each pancreas were microscopically examined for infiltrating mononuclear cells.
Thymus immunohistology and confocal microscopy.
Thymi from ∼12-week-old mice were frozen in OCT compound (TissueTek), sectioned (8–10 μm), and stained, as previously described (17), with the following primary antibodies: polyclonal rabbit anti-cytokeratin (DakoCytomation), anti-Aire (clone 5H12, ), ER-TR7 (detects reticular fibroblasts, ), and MTS24 (detects an embryonic thymic population containing epithelial progenitor cells and a subset of medullary epithelium in adult thymi, ). These were detected with Alexa Fluor 647–conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 568–conjugated goat anti-rat IgG (H+L) (Molecular Probes). For three-color immunofluorescence, sections were blocked with 10% (vol/vol) normal rat serum then stained with biotinylated anti-CD19 (clone 1D3; Pharmingen) or biotinylated MTS10 (detects medullary epithelium ) and streptavidin Alexa Fluor 488–conjugated anti-rat IgG (H+L) (Molecular Probes). Sections were mounted with fluorescent mounting medium (DakoCytomation), and images were acquired on a Bio-Rad MRC 1024 confocal microscope with a Kr/Ar laser (excitation lines 488, 568, and 647 nm).
Thymic stromal cell isolation and flow cytometry.
Thymic stromal cell isolation from ∼12-week-old mice was performed as previously described (26). Multiparameter flow cytometric analysis of the final digests, enriched for thymic stromal cells, was performed using biotinylated anti-Ly51 (clone 6C3; Pharmingen), biotinylated UEA1 lectin (Vector), phycoerythrin-conjugated anti-CD45 (clone 30-F11; Pharmingen), biotinylated anti-EpCAM (Clone G8.8a; a generous gift from A. Farr), MTS15 (detects reticular fibroblasts at the blood-thymus barrier), and MTS24. Secondary reagents used were allophycocyanin-conjugated streptavidin and fluorescein isothiocyanate–conjugated anti-rat IgG (H+L) (Molecular Probes). A 10% normal rat serum was used to prevent crossreactive binding of antibodies following fluorescein isothiocyanate–conjugated anti-rat IgG staining. FACScalibur and CellQuest software (Becton Dickinson) were used for flow cytometric analysis, with exclusion of nonviable and hemopoietic cells based on propidium iodide incorporation and CD45 expression.
Flow cytometry analysis of NKT cells.
Cell suspensions of thymus, spleen, and liver were prepared from 5- to 6-week-old mice, as previously described (27). The following monoclonal antibodies were used in multiparameter flow cytometric analysis: anti-B220-FITC (clone RA3–6B2), anti-CD4-APC and PerCP (clone RM4–5), anti-CD8-PerCP (clone 53–6.7), anti-CD25-PE (clone PC61), and anti-TCRβ-APC (clone H57–597) (all purchased from PharMingen, San Diego, CA). Phycoerythrin-conjugated, α-galactsylceramide (a-GalCer)-loaded mCD1d tetramers were produced in-house as described (27) using recombinant baculovirus encoding his-tagged mouse CD1d and β-2 microglobulin (kindly provided by M. Kronenberg’s laboratory, Division of Developmental Immunology, La Jolla Institute of Allergy and Immunology, San Diego, CA). Fc-receptor block (anti-CD16/CD32, clone 2.4G2, grown in-house) was added to all staining cocktails. Monoclonal antibodies were purchased from BD PharMingen. One fluorescence channel was typically not used for specific staining but instead used for the exclusion of autofluorescent cells. Flow cytometry was performed using a FACSCalibur (Becton Dickinson) and analyzed using CELLQuest software (Becton Dickinson). Quantitative difference between two samples were compared with the Mann-Whitney U (rank-sum) test.
While this manuscript was under review, an article was published that provided additional evidence for the presence of a diabetes susceptibility locus within a region on Chr4 encompassing the Idd11 interval defined here (1).
Additional information for this article can be found in an online appendix available at http://diabetes.diabetesjournals.org.
This work was supported by the Juvenile Diabetes Research Foundation, the Cooperative Research Centre for the Discovery of Genes for Common Human Diseases, and the National Health and Medical Research Council. D.I.G. is supported by a National Health and Medical Research Council of Australia research fellowship.
The authors thank Maya Kesar and Davina Radford for assistance with animal care and Timothy Williams for technical assistance. All experiments described within this text were performed in Australia and comply with the current laws of Australia regarding such experiments.