Dysregulated liver lipid metabolism and innate immunity associated with hepatic steatosis in neonatal BBdp rats and NOD mice.

In a previous study we reported that prediabetic rats have a unique gene signature that was apparent even in neonates. Several of the changes we observed, including enhanced expression of pro-inflammatory genes and dysregulated UPR and metabolism genes were first observed in the liver followed by the pancreas. In the present study we investigated further early changes in hepatic innate immunity and metabolism in two models of type 1 diabetes (T1D), the BBdp rat and NOD mouse. There was a striking increase in lipid deposits in liver, particularly in neonatal BBdp rats, with a less striking but significant increase in neonatal NOD mice in association with dysregulated expression of lipid metabolism genes. This was associated with a decreased number of extramedullary hematopoietic clusters as well as CD68+ macrophages in the liver of both models. In addition, PPARÉ£ and phosphorylated AMPKα protein were decreased in neonatal BBdp rats. BBdp rats displayed decreased expression of antimicrobial genes in neonates and decreased M2 genes at 30 days. This suggests hepatic steatosis could be a common early feature in development of T1D that impacts metabolic homeostasis and tolerogenic phenotype in the prediabetic liver.

An orthologous non-MHC locus in rats and mice is linked to CD4+ and CD8+ T-cell proportion.

CD4+ and CD8+ T cells have a central role in the immune system due to their ability to protect against infection and cancer development without targeting self. Consequently, changes in CD4+ and CD8+ T-cell homeostasis can be indicative of an array of serious illnesses, ranging from viral infections to autoimmune diseases. In addition to environmental influences, there is evidence for a genetic component regulating the proportion of CD4+ and CD8+ T cells in lymphoid organs. Indeed, identifying the genetic determinants defining the frequency of the T-cell subsets is critical as it may reveal a targetable genetic pathway to modulate CD4+ and CD8+ T-cell numbers, which could be of clinical relevance for multiple disease settings. In this study, we aim to uncover non-MHC genetic factors regulating the proportion of CD4+ and CD8+ T cells in lymphoid tissues. By investigating linkage analyses on three independent F2 cohorts, namely a rat F2 (BBDP × ACI.1U.LYP) cohort, a mouse 3A9 TCR transgenic F2 (B10.BR × NOD.H2k) cohort and a mouse F2 (C57BL/6 × FVB/N) cohort, we uncover an orthologous non-MHC locus on rat chromosome 1 and mouse chromosome 7 that is linked to T-cell proportion amongst total lymphocytes.

Genetic dissection of Iddm26 in the spontaneously diabetic BBDP rat.

The 40 Mb T1D susceptibility locus Iddm26 was mapped to chromosome 2 through linkage analysis of a conditioned cross-intercross between the diabetes-prone BBDP and the diabetes-resistant ACI.BBDP-Iddm1,Iddm2 (ACI.1u.Lyp). It is flanked by Iddm32 and Iddm33, which control the kinetics of disease progression. To fine-map Iddm26 and characterize immune phenotypes controlled by this locus, several congenic sublines were generated carrying smaller, overlapping intervals spanning Iddm26 and fragments of Iddm32 and 33. Analysis of disease susceptibility, age of disease onset, and immune phenotypes in these sublines identified subloci regulating these different parameters. Two ACI.1u.Lyp-derived subloci, Iddm26.1 and Iddm26.2, imparted significant protection from diabetes, decreasing the cumulative incidence by as much as 57% and 28%, respectively. Iddm26.2, which overlaps with the human PTPN22 locus, only affected disease susceptibility, whereas Iddm26.1 also significantly affected disease kinetics, delaying T1D onset by more than 10 days compared with the parental BBDP strain. These Iddm26 subloci also regulated various immune phenotypes, including the proportion of splenic macrophages by Iddm26.1, and the proportion of activated T-cells in secondary lymphoid organs by Iddm26.2. The analysis of Iddm26 congenic animals in two different SPF facilities demonstrated that the influence of this locus on T1D is environment-dependent.

BB rat lyp mutation and Type 1 diabetes.

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.

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.

Kinetics of T cell turnover following thymectomy.

We develop a mathematical formula that provides the number of cells in an isolated population that have divided k times in n days (O< or =k< or =n). This differential cell division formula is applied to the kinetics of peripheral T cells in the diabetes-prone BB rat following thymectomy. These rats received daily intraperitoneal injections of the DNA precursor, bromodeoxyuridine (BrdUrd), over a period of 12-13 days. As the cells divided, they incorporated BrdUrd progressively into their DNA molecules, and the differential formula provides a close prediction of the fraction of BrdUrd-positive T cells present each day during this 'pulse' phase. No further BrdUrd was administered after 13 days, and the diminishing fraction of BrdUrd-positive cells was recorded for several more weeks. The differential cell division formula was capable of describing the rather complex form of the retention curve as BrdUrd-tagged DNA molecules passed to progeny cells during this 'chase' phase. We believe that this simple formula may be found generally useful in describing cell kinetic data in mitotically active cells.

Speculation on the lineage relationships among CD4(-)8(+) gut-derived T cells and their role(s).

The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been established. Cryptopatches have now been identified as the location of the elusive precursors for gut-derived T cells. These cryptopatch cells have been shown to give rise to intestinal T cells expressing either TCRgammadelta or TCRalphabeta. Here we discuss the role of MHC in the development and selection of gut-derived T cells. Through the analysis of iIEL selection in animals expressing a transgenic TCRalphabeta, in the presence or absence of p56(lck), we discuss lineage relationships among CD4(-)8(+) iIEL subsets, and their possible function(s).

T cell reconstitution of BB/W rats after the initiation of insulitis precipitates the onset of diabetes.

One of the diabetes susceptibility genes of the BB/W (Biobreeding/Worcester) rat maps to the lyp locus on chromosome 4. The BB/W lyp allele is responsible for a severe peripheral T lymphopenia. Correction of this lymphopenia by transfer of normal, histocompatible T cells prevents diabetes, providing T cell reconstitution is initiated before insulitis. We have analyzed this time-dependent regulation of the diabetogenic process by normal T cells. We demonstrate that T cell reconstitution after the initiation of insulitis precipitates the onset of diabetes through the recruitment of donor T cells to the autoimmune process. This inability of normal T cells to regulate primed diabetogenic BB/W T cells and their own autoreactive potential were observed when normal T cells outnumbered pathogenic T cells by approximately 1000-fold. Analysis of donor-derived T cells recovered from BB/W rats that were reconstituted before insulitis, and hence protected from diabetes, demonstrates that early T cell reconstitution of BB/W rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among donor cells or in the expansion of T cells that can regulate the activation and expansion of diabetogenic T cells.

Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat.

One of the diabetes susceptibility genes of the BB rat is a mutation at the lyp locus that decreases the thymic output of T cells and the life span of most recent thymic emigrants (RTE). Consequently, there is a 10-fold reduction in the number of CD4+ and CD8+ T cells in secondary lymphoid organs. Results presented in this work demonstrate that the BB rat lyp mutation is associated with an accelerated apoptotic death in vitro of mature CD4+ 8- and CD4- 8+ thymocytes and peripheral T cells. The stability of the pool of recirculating T cells (PRL) of BB rats over time results from a > 10-fold increase in the mitotic activity of T cells as assessed in vivo by bromodeoxyuridine incorporation. This increased mitotic activity is not observed when BB T cells develop in the context of a normal sized PRL. MHC haploidentical WF and BB rats differ at minor histocompatibility loci. Intravenous injection of (WF x BB)F1 T cells into euthymic BB rats led to the rejection of donor T cells within 3 wk by unprimed recipients and within 1 wk by primed recipients. This secondary immune response was unaffected by postpriming thymectomy. F1 T cells were not rejected, but rather expanded after their injection into thymectomized BB rats that had been primed as early as 48 h after thymectomy. These results strongly suggest that the BB rat PRL is devoid of long-lived naive T cells and that rescue of recent thymic emigrants from programmed cell death is initiated by Ags, exclusively.

Excessive production of nitric oxide by macrophages from DP-BB rats is secondary to the T-lymphopenic state of these animals.

Activated macrophages are the first mononuclear cells to migrate to the pancreas of DP-BB rats at the initiation of insulitis. These cells produce an excess of NO, which has been implicated as a mediator of both beta-cell damage and inhibition of T-cell proliferation in this rat strain. Genetic studies have shown that the impaired proliferative response of T-cells segregates with one of the diabetes-susceptibility genes of the DP-BB rat, lyp, which is responsible for a peripheral T-lymphopenia. This observation suggests that the dysregulated expression of inducible NO synthase (iNOS) is under the control of lyp itself or a gene in linkage disequilibrium with lyp. Using two models of hemopoietic chimeras--DP-BB rats reconstituted with isocongenic T-cells and irradiated (WF x DP-BB)F1 animals reconstituted with bone marrow of both parental strains--we demonstrated that the production of NO by DP-BB macrophages is normal when these cells originate from a non-T-lymphopenic environment. Consequently, these macrophages no longer inhibit the stimulation of DNA synthesis in activated T-cells. Macrophages of young WF rats were found to produce high levels of NO, which inhibited T-cell proliferation in vitro. This observation strongly suggests that upregulation of NO synthesis in DP-BB macrophages represents the abnormal persistence of a phenomenon restricted to the first few weeks of life in non-diabetes-prone rats. Taken together, these results demonstrate that the elevated production of NO by DP-BB macrophages results from the lyp mutation and represents a crucial mechanism through which T-lymphopenia contributes to the development of diabetes.

T-cell development and selection in the intestinal epithelium.

Though it is now well established that T lymphocytes develop in the murine intestinal epithelium, many features of T lymphopoiesis occurring at this site remain controversial and poorly understood. One of the most contentious issues is whether all TcR alpha beta+ iIEL subsets, characterized by the differential expression of CD4, CD8 alpha and CD8 alpha, develop in situ, as suggested by the analysis of athymic radiation chimeras and parabionts. Athymic radiation chimeras have also been used to study positive and negative selection events imposed on developing iIEL expressing an MHC class I restricted transgenic TcR alpha beta. Results reveal that while distinct mechanisms may regulate these processes in the gut, the functional repertoire generated at this site is identical to that resulting from intra-thymic T-cell development. Strikingly, this is not the case for the development and selection of iIEL expressing an MHC class II restricted TcR alpha beta. Features peculiar to MHC class II molecules expressed by enterocytes are discussed in this context. In addition, two model systems are presented towards understanding the basis for the observed oligoclonal repertoire of TcR alpha beta+ iIEL. Specifically, the role of luminal antigen is addressed by comparing the repertoire of iIEL developing in the orthotopic gut with that of iIEL developing in an ectopic sterile intestine in the same athymic animal. The role of TcR alpha beta-mediated signals in support of the putative oligoclonal expansion of iIEL, is addressed using mice lacking the protein tyrosine kinase, Lck.

Glucose turnover after a mixed meal in dogs: glucoregulation without change in arterial glycemia.

Because the dog can respond to a mixed-meal challenge with little or no change in plasma glucose concentration, we used kinetic techniques to quantify the magnitude and duration of changes in glucoregulation. Glucose turnover was measured using [3-3H]glucose and [U-14C]glucose over two 19-h periods in healthy dogs, first during a fast (n = 6) and then throughout the postprandial state (n = 6) after a single mixed meal. Mean arterial glycemia remained constant in the fasted state (7.5 +/- 0.2 mM) and in the fed state (7.6 +/- 0.3 mM). Glucose appearance (Ra), however, increased slowly after the meal from 38 +/- 2 mg/min to a maximum of 79 +/- 8 mg/min after 6 h and stayed elevated until 12 h (P < 0.001). In parallel, glucose disappearance (Rd) rose from 35 +/- 3 to 83 +/- 7 mg/min, closely matching the corresponding Ra. Glucose recycling rose from 25 +/- 8% at baseline to a maximum of 53 +/- 15% (P < 0.05) at 14 h in fed dogs, whereas levels for fasted dogs stayed between 19 +/- 7% at 0 h and 27 +/- 12% at 6 h. Insulin levels rose significantly 30 min after the meal from 67 +/- 7 pM to a peak of 208 +/- 54 pM at 6 h but remained elevated for 12 h. We conclude that 1) the dog was able to maintain postprandial glucoregulation by very precise matching of Ra and Rd such as to maintain glycemia constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymus independent T cell development and selection in the intestinal epithelium.

The presence of intestinal intra-epithelial lymphocytes (IEL) has been appreciated for over 100 years. However, until recently, the IEL were thought to be simply another specialized component of the gut-associated lymphoid tissue (GALT), sharing the same origin as lymphocytes found in other GALT compartments such as the lamina propria, the Peyer's patches, and the mesenteric lymph nodes. While the IEL remain functionally enigmatic, over the last ten years increasing evidence, originally based on the phenotypic complexity of IEL, has led to the conclusion that the intestinal epithelium comprises a distinct and unique lymphoid compartment within the organism. The recent discovery that the intestinal epithelium is a primary T lymphopoietic organ provides obvious potential explanations for the phenotypic peculiarities of IEL. The following discussion reviews the experiments that established that the intestinal epithelium is a site of T cell development and selection, and the discussion correlates phenotypic and functional heterogeneity of IEL within this context.

Thymus-independent positive and negative selection of T cells expressing a major histocompatibility complex class I restricted transgenic T cell receptor alpha/beta in the intestinal epithelium.

We demonstrate that in the mouse intestinal epithelium the selection of T lymphocytes expressing a transgenic T cell receptor alpha/beta (TCR-alpha/beta) specific for male antigen (H-Y) in the context of H-2Db depends on the differential expression of H-Y and H-2Db in situ. In H-2Db transgenic males, there is no reduction in the number of intestinal intraepithelial lymphocytes (IEL), and the four main subsets of IEL expressing TCR-alpha/beta, defined by the differential expression of CD4, CD8 alpha, and CD8 beta, are present. Moreover, the level of expression of CD8 alpha and CD8 beta on CD8+ IEL subsets is unaltered. The frequency of CD8 alpha+ IEL expressing CD8 beta, in H-2Db male mice, however, is significantly decreased and these cells do not express the transgenic TCR. In contrast, virtually all CD8 alpha+beta- IEL in the same animals express the transgenic TCR. Still, these potentially autoreactive cells are refractile to H-Y/H-2Db stimulation in vitro. Both H-2Db and H-2Dd transgenic females contain high frequencies of cells expressing the transgenic TCR among CD8 alpha+beta- and CD8 alpha+beta+ IEL. However, two possibly related phenotypic features are peculiar to H-2Db female mice. The frequency of CD8 alpha+ IEL expressing CD8 beta is increased in these mice and, while in H-2Dd females the level of the transgenic TCR alpha chain expressed on CD8 alpha+beta+ IEL is uniformly low, some of the CD8 alpha+beta+ IEL in H-2Db females express a high level of both transgenic TCR chains. It is important to note, the ability of CD8 alpha+beta+ IEL to respond to H-Y/H-2Db stimulation in vitro is restricted to those coexpressing a high level of both transgenic TCR chains. The analysis of athymic radiation chimeras using adult thymectomized recipients of distinct H-Y/H-2 haplotypes, reconstituted with bone marrow from H-2Db transgenic females, demonstrates that all IEL subsets present in unmanipulated transgenic animals develop in the absence of a thymus. These IEL are phenotypically identical to those found in unmanipulated transgenic animals sharing the H-Y/H-2 haplotype of athymic recipients. Taken together, these results demonstrate that in the absence of male antigen, expression of H-2Db in the intestinal epithelium results in the positive selection of functional IEL specific for male antigen, in situ. When both H-Y and H-2Db are expressed in the intestinal epithelium, CD8 alpha+beta+ IEL expressing the transgenic TCR are negatively selected, while the frequency of nonfunctional CD8 alpha+beta- IEL expressing the transgenic CR is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Different rates of conversion to IDDM in siblings of type 1 diabetic children: the Montreal family study.

This study was undertaken to analyze the risk of developing insulin-dependent diabetes (IDDM) in siblings of type 1 diabetic children. Islet-cell antibodies (ICA) were tested in 568 subjects, siblings of type 1 diabetic children. The subjects were followed prospectively for the conversion to clinical diagnosis of IDDM. As a result siblings who were islet cell antibody (ICA)+ positive at the time of diagnosis of the diabetic sibling (index case) had a significantly higher risk of developing IDDM than those who were ICA-. However, of the 19 siblings who developed IDDM, only 10 were ICA+ at the time of the first test but, 17 became ICA+ before diagnosis of IDDM. The interval between a positive test and the clinical diagnosis of IDDM varied between subjects (6-44 months, mean = 18.4 +/- 4.2 S.E. months) but it was less than 1 year in one subject. In addition to the higher risk of developing IDDM when ICA was positive, male sex and younger age of the subjects as well as young age of the index case and multiplex pedigrees were significant predictors of conversion to IDDM. The Cox's regression tree constructed using RECPAM identified three groups of varying rates of conversion to IDDM: (1) a group with the slowest progression characterized by ICA- and age of index case > 5 years or female sex (relative hazard = 1); (2) an intermediate progression group consisting of subjects who are ICA- and have both < 5 years of age and male sex (relative hazard = 8.78); (3) a group with the fastest progression consisting of subjects who are ICA+ (relative hazard = 31.45). From these results our data suggest that in addition to ICA, clinical markers such as age, sex and multiple pedigrees are also significant predictors of the rate of conversion to IDDM. Furthermore, screening for ICA in family intervention studies will have to be done frequently, perhaps yearly, and will have to be continued into adult life, particularly in ICA- subjects in order to identify the 85-90% of subjects who become ICA+ at the clinical diagnosis of IDDM.

CD8+ T-cells are required for adoptive transfer of the BB rat diabetic syndrome.

LGLs with NK activity account for the majority of BB rat PBLs expressing CD8, and it has been suggested that these LGL/NK cells are involved in the pathogenesis of the BB rat diabetic syndrome. By using a recently developed mouse MoAb, 3.2.3, specific for rat LGL, we demonstrate that BB and WF rat LGLs are phenotypically and functionally similar. To directly assess the role of LGLs in the development of diabetes in vivo, an adoptive transfer of T-cells to young LGL/NK cell-depleted diabetes-prone BB rats was performed. CD4+8- and CD4-8+ T-cells (> 98.5% pure), isolated from diabetic BB rats, were activated in vitro and injected into 30-day-old diabetes-prone BB rats. Recipients were either chronically injected with 3.2.3 (n = 15) or received an isotype-matched irrelevant MoAB (n = 14). Secondary lymphoid organs of 3.2.3-treated recipients contained < 0.1% 3.2.3+ lymphocytes, and this depletion was associated with a major decrease in the NK activity of their splenocytes. Despite this, the incidence of diabetes in 3.2.3-treated animals (40%) was not significantly different from that observed in control recipients (57%). Thus, the BB rat diabetic syndrome can be adoptively transferred in the absence of LGL/NK cells, suggesting that BB rat CD8+ T-cells are involved in the diabetogenic process. To assess the pathogenic role of CD8+ T-cells, we compared the incidence of diabetes in three groups of diabetes-prone BB recipients after injection of T-cells isolated from diabetic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Polygenic nature of spontaneous diabetes in the rat. Permissive MHC haplotype and presence of the lymphopenic trait of the BB rat are not sufficient to produce susceptibility.

We describe the phenotypic characteristics of animals in the fifth backcross-intercross generation of a breeding program in which the RT1 u haplotype and the phenotypic trait responsible for the T-lymphopenia of BB rats have been transferred to the ACI background. In this generation of animals, 24% were lymphopenic with decreased numbers of PBL expressing CD5, TCR alpha, and RT6. The PBL of the lymphopenic animals had a decreased mitogenic response to ConA. All of the nonlymphopenic animals were homozygous for RT6.2. Phenotypic analysis of intestinal IEL revealed that this was also the case for the lymphopenic animals. Moreover, IEL of the lymphopenic animals exhibited a pattern of staining (increased numbers of TCR alpha beta+CD4+CD8+ and decreased numbers of TCR alpha beta+CD4-CD8+) similar to that of BB DP animals. The ACI.1U(BB)-lymphopenic animals, although having two of the genetic traits associated with the expression of spontaneous diabetes mellitus, uniformly fail to develop diabetes. Breeding studies in which these animals were crossed with BB and hBB rats suggest that other genes are necessary for development of overt diabetes.

Factors predicting course of beta-cell function in IDDM.

OBJECTIVE: The purpose of this study was to determine whether the severity o clinical presentation, sex, age, HLA type, and the presence of IAs and ICAs could predict the variation of residual insulin secretion as measured by the serum C-peptide response to a Sustacal meal. RESEARCH DESIGN AND METHODS: A cohort of 151 newly diagnosed IDDM children (mean age 10.2 +/- 4.6 yr) was followed prospectively for 3 yr. Thirty-five patients (12 males, 23 females) were still secreting C-peptide after 36 mo. RESULTS: We found that age (P = 0.0001), sex (P = 0.003), presence of ICA (P = 0.006), severity of clinical presentation (P = 0.001), and symptom duration (P = 0.002) significantly predicted the rate of loss of C-peptide secretion. The risks of accelerated C-peptide disappearance decreased with increasing age, the risk ratios being 0.25 for the older group (greater than 12 yr) compared with the younger group (less than 6 yr) and 0.50 for the intermediate group (6-12 yr) compared with the younger group. The risk for the presence of ICA was 1.7, and the risk for males was 1.7 also. There was a significant negative correlation between ICA titers and C-peptide at 18 and 24 mo after diagnosis (P = 0.04). There were no significant differences in HbA1 values between patients who secreted C-peptide and those who did not. CONCLUSIONS: We conclude that younger age of onset, male sex, high titers of ICA, severe clinical presentation, and shorter symptom duration significantly predict accelerated rates of loss of C-peptide secretion.