Philippe Poussier

The diabetic syndrome of the ‘BB’ Wistar rat: Possible relevance to Type 1 (insulin-dependent) diabetes in man

SummaryThe diabetes which occurs spontaneously in the ‘BB’ Wistar rat has many affinities with human Type 1 (insulin-dependent) diabetes. It occurs in a non-obese, standard, laboratory rat derived from a non-inbred Wistar line. Both sexes are affected, with onset corresponding approximately to the time of sexual maturation. Both genetic and immune factors are involved in the aetiology, but their precise nature remains to be defined. Evolution of the overt clinical syndrome occurs over a period of hours to a few days. An intense insulitis is found, accompanied by selective destruction of B cells. Although insulitis may precede diabetes by many weeks, within 7–21 days after glycosuria the B cells are completely destroyed and have disappeared and the islets are few, small and with little residual inflammation. If untreated, marked wasting of body tissues, including fat and muscle protein, dehydration, and ketosis supervene. Careful study of littermates reveals glucose intolerance in 10%–25%, accompanied always by insulitis and these rats may subsequently develop insulin-dependent diabetes. Marked lymphopenia, mainly of thymus-derived (T) lymphocytes, both precedes and is sustained during glucose intolerance and overt diabetes. This lymphopenia appears to be associated reliably with insulitis, and may be a simple marker of susceptibility thereto. Abnormalities of nerves, testicles, and a tendency towards increased frequency of lymphomas have been found. Further research in this animal could lead to insights into aetiology, pathophysiology and complications potentially applicable to man.

A unique subset of self-specific intraintestinal T cells maintains gut integrity.

Lymphocytes residing in the intestinal epithelium are exclusively T cells and account for one of the largest collection of T cells in the organism. However, their function remains obscure. We and others have shown that the development of intestinal intraepithelial T cells is compromised in mutant mice prone to chronic intestinal inflammation. These results led us to directly assess their role in regulating the development of colitis secondary to transfer of primary splenic TCRαβ+CD4+CD45RBhi T cells into severe combined immunodeficiency (SCID) mice. Here we demonstrate that prior reconstitution of SCID recipients with intraintestinal TCRαβ+CD4−CD8α+β− T cells prevents disease, and does so in an interleukin (IL)-10–dependent fashion. In contrast, reconstitution with either TCRγδ+ or TCRαβ+CD4− CD8α+β+ intestinal T cells did not prevent colitis. TCRαβ+CD4−8α+β− T cells are unique to the intestinal epithelium of both rodents and humans. Previous repertoire analyses of TCRαβ+CD4−CD8α+β− T cells revealed a high proportion of cells expressing high affinity, self-specific TCR within this subset. We demonstrate that monoclonal, self specific TCRαβ+CD4−CD8α+β− cells derived from TCR transgenic mice also prevent the onset of colitis. Thus, intestinal TCRαβ+CD4−CD8α+β− T cells, selected based on their self-reactivity, maintain gut integrity in a IL-10–dependent fashion.

Islet Cell Surface Antibodies and Lymphocyte Antibodies in the Spontaneously Diabetic BB Wistar Rat

Plasma from 14 diabetic and 6 nondiabetic BB Wistar rats along with plasma from 6 non-BB Wistar rats was evaluated for the presence of islet cells surface antibodies (ICSA) and antibodies to spleen lymphocytes by the protein-A radioligand assay. Dispersed Wistar rat islet cells incubated with plasma from diabetic rats bound 4255 ± 2208 cpm 125I-protein A/5 × 104 islet cells (mean ± SD) compared with 984 ± 454 cpm/5 × 104 islet cells in islet cells incubated with plasma from nondiabetic BB rats (P < 0.005). Twelve of the 14 diabetic rats with a duration of diabetes for 3–11 days bound radioactivity above the mean and 2 × SD of controls. The binding of 125I-protein A did not differ between nondiabetic BB rats and non-BB Wistar rats. Wistar rat spleen lymphocytes incubated in diabetic plasma bound 20,249 ± 10,783 cpm/2 × 106 spleen lymphocytes compared with 3460 ± 1809 in the controls (P < 0.005). Animals positive for ICSA correlated with those positive for spleen lymphocyte antibodies. It is concluded that ICSA and splenic lymphocyte antibodies are present in diabetic BB rats.

Impaired Post-Thymic Development of Regulatory CD4+25+ T Cells Contributes to Diabetes Pathogenesis in BB Rats

One of the BB rat diabetes (diabetes mellitus (DM)) susceptibility genes is an Ian5 mutation resulting in premature apoptosis of naive T cells. Impaired differentiation of regulatory T cells has been suggested as one possible mechanism through which this mutation contributes to antipancreatic autoimmunity. Using Ian5 congenic inbred rats (wild-type (non-lyp BB) and mutated (BB)), we assessed the development of BB regulatory CD8−4+25+T cells and their role in the pathogenesis of DM. BB rats have normal numbers of functional CD8−4+25+Foxp3+ thymocytes. The proportion of CD25+ cells among CD8−4+ recent thymic emigrants is also normal while it is increased among more mature CD8−4+ T cells. However, BB CD8−4+25+Foxp3+ thymocytes fail to undergo homeostatic expansion and survive upon transfer to nude BB rats while Foxp3 expression is reduced in mature CD8−4+25+ T cells suggesting that these cells are mostly activated cells. Consistent with this interpretation, peripheral BB CD8−4+25+ T cells do not suppress anti-TCR-mediated activation of non-lyp BB CD8−4+25− T cells but rather stimulate it. Furthermore, adoptive transfer of unfractionated T cells from diabetic BB donors induces DM in 71% of the recipients while no DM occurred when donor T cells are depleted of CD8−4+25+ cells. Adoptive transfer of 106 regulatory non-lyp BB CD8−4+25+ T cells to young BB rats protects the recipients from DM. Taken together, these results demonstrate that the BB rat Ian5 mutation alters the survival and function of regulatory CD8−4+25+ T cells at the post-thymic level, resulting in clonal expansion of diabetogenic T cells among peripheral CD8−4+25+ cells.

TYPE 1 DIABETES IN THE BB RAT: A POLYGENIC DISEASE

OBJECTIVE Two type 1 diabetes susceptibility genes have been identified in the spontaneously diabetic biobreeding diabetes-prone (BBDP) rat, the major histocompatibility complex (MHC) (RT1) class II u haplotype (Iddm1) and Gimap5 (Iddm2). The strong effects of these have impeded previous efforts to map additional loci. We tested the hypothesis that type 1 diabetes is a polygenic disease in the BBDP rat. RESEARCH DESIGN AND METHODS We performed the most comprehensive genome-wide linkage analysis for type 1 diabetes, age of disease onset (AOO), and insulitis subphenotypes in 574 F2 animals from a cross-intercross between BBDP and type 1 diabetes–resistant, double congenic ACI.BBDP-RT1u,Gimap5 (ACI.BB1u.lyp) rats, where both Iddm1 and Iddm2 were fixed as BBDP. RESULTS A total of 19% of these F2 animals developed type 1 diabetes, and eight type 1 diabetes susceptibility loci were mapped, six showing significant linkage (chromosomes 1, 3, 6 [two loci], 12, and 14) and two (chromosomes 2 and 17) suggestive linkage. The chromosomes 6, 12, and 14 intervals were also linked to the severity of islet infiltration by immunocytes, while those on chromosomes 1, 6 (two loci), 14, 17, and a type 1 diabetes–unlinked chromosome 8 interval showed significant linkage to the degree of islet atrophy. Four loci exhibited suggestive linkage to AOO on chromosomes 2 (two loci), 7, and 18 but were unlinked to type 1 diabetes. INS, PTPN22, IL2/IL21, C1QTNF6, and C12orf30, associated with human type 1 diabetes, are contained within the chromosomes 1, 2, 7, and 12 loci. CONCLUSIONS This study demonstrates that the BBDP diabetic syndrome is a complex, polygenic disease that may share additional susceptibility genes besides MHC class II with human type 1 diabetes.

BB rat lyp mutation and Type 1 diabetes

Summary: BioBreeding (BB) rats spontaneously develop an autoimmune diabetic syndrome similar to that observed in humans and NOD mice. One of the diabetes susceptibility loci maps to the lyp locus on chromosome 4. In this article we describe the consequences of the BB rat lyp mutation on T‐cell homeostasis, repertoire and function, as well as its role in the pathogenesis of type I diabetes.

Suppressor of Cytokine Signaling 1 Regulates IL-15 Receptor Signaling in CD8+CD44high Memory T Lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1−/−IFN-γ−/− mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44high memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rβ, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44high T cells from SOCS1−/−IFN-γ−/− mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-α production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-xL in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common γ-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.

Islet cell surface and lymphocyte antibodies often precede the spontaneous diabetes in the BB rat

SummaryThe diabetic syndrome of the BB rat shows many homologies with that of human insulin-dependent diabetes and evidence that the onset of the disease is associated with the presence of autoantibodies, including islet cell surface antibodies. In this study, sera were sampled serially from weaning to 157 days of age from 26 BB rats in two low-incidence litters, and 22 rats of three high-incidence litters. Clinical and metabolic variables were monitored concurrently with blood lymphocyte counts. Islet morphology was correlated at sacrifice. In the high-incidence litters, eight rats developed insulin-dependent diabetes, five impaired glucose tolerance, and the remaining nine all showed insulitis. In the low-incidence litters, only one animal showed impaired glucose tolerance and another insulitis. In the high-incidence litters 16 rats (73%) had islet cell surface antibodies compared with 4 out of 26 (15%) low-incidence controls (p<0.002). Antibodies reactive with Wistar rat spleen lymphocytes were present in all high-incidence rats compared with 19% (5 out of 26) among the control litters (p<0.002). Time courses of islet cell surface and lymphocyte antibody appearance and their peak values varied, but already at weaning the levels of both antibodies were increased among the high-incidence litter rats (p< 0.001). Islet cell surface and/or lymphocyte antibodies were therefore present in the majority of animals at an age where neither morphological nor metabolic evidence of the diabetic syndrome were yet detected. All rats that showed any form of the syndrome were lymphopenic. These findings suggest that BB rats have an abnormal immune response which predisposes to later development of insulin-dependent diabetes, often preceded by the presence of islet cell surface and/or lymphocyte antibodies.

Intestinal inflammation observed in IL-2R/IL-2 mutant mice is associated with impaired intestinal T lymphopoiesis

BACKGROUND & AIMS Although interleukin (IL)-2(-/-) and IL-2Ralpha(-/-) mice develop inflammatory bowel disease, IL-2Rbeta(-/-) animals are apparently free of gut pathology. Intraintestinal T lymphopoiesis is reported to be impaired in IL-2Rbeta(-/-) mice; we have determined whether this characteristic correlated with the apparent resistance of this mutant strain to intestinal inflammation. This led us to reassess intraintestinal T lymphopoiesis in these 3 mutant strains. METHODS Intestinal histology and intraintestinal T lymphopoiesis were analyzed in unmanipulated mutant mice and in athymic and euthymic radiation chimeras reconstituted with bone marrow derived from IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) donors. RESULTS Intraintestinal T lymphopoiesis was ablated in the 3 mutant strains and was associated with cryptopatch abnormalities. The intestinal mucosa of mice reconstituted with lymphocytes from IL-2Rbeta(-/-) mice exhibited lesions of both the small and large bowel similar to those observed in the early stages of human gluten enteropathy and acute ulcerative colitis, respectively. Analysis of euthymic and athymic bone marrow radiation chimeras indicated that T cells located in the intestinal mucosa of unmanipulated IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) mice are of thymic origin. CONCLUSIONS Null mutations at IL-2/IL-2Ralpha and beta loci differentially affect intraintestinal and intrathymic T lymphopoiesis. These conditions are associated with lesions of intestinal inflammation that are mediated by thymus-derived T cells.

Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics.