Diubiquitin (Ubd) is a susceptibility gene for virus-triggered autoimmune diabetes in rats.

Genetic studies of type 1 diabetes (T1D) have been advanced by comparative analysis of multiple susceptible and resistant rat strains with a permissive class II MHC haplotype, RT1(u). LEW.1WR1 (but not resistant LEW.1W or WF) rats are susceptible to T1D induced by a TLR3 agonist polyinosinic:polycytidylic acid followed by infection with parvovirus. We have mapped genetic loci for virus-induced T1D susceptibility, identifying a major susceptibility locus (Iddm37) near the MHC. The Iddm37 homologs on mouse and human chromosomes are also diabetes linked. We report that a major effect gene within Iddm37 is diubiquitin (Ubd). Gene expression profiling of pancreatic lymph nodes in susceptible and resistant rats during disease induction showed differences in Ubd transcript abundance. The LEW.1WR1 Ubd promoter allele leads to higher inducible levels of UBD than that of LEW.1W or WF. Using zinc-finger nucleases , we deleted a segment of the LEW.1WR1 Ubd gene and eliminated its expression. UBD-deficient rats show substantially reduced diabetes after viral infection. Complementary studies show that there may be another diabetes gene in addition to Ubd in the Iddm37 interval. These data prove that Ubd is a diabetes susceptibility gene, providing insight into the interplay of multiple genes and environmental factors in T1D susceptibility.

Temporal induction of immunoregulatory processes coincides with age-dependent resistance to viral-induced type 1 diabetes.

The dilute plasma cytokine milieu associated with type 1 diabetes (T1D), while difficult to measure directly, is sufficient to drive transcription in a bioassay that uses healthy leukocytes as reporters. Previously, we reported disease-associated, partially IL-1 dependent, transcriptional signatures in both T1D patients and the BioBreeding (BB) rat model. Here, we examine temporal signatures in congenic BBDR.lyp/lyp rats that develop spontaneous T1D, and BBDR rats where T1D progresses only after immunological perturbation in young animals. After weaning, the BBDR temporal signature showed early coincident induction of transcription related to innate inflammation as well as IL-10- and TGF-ß-mediated regulation. BBDR plasma cytokine levels mirrored the signatures showing early inflammation, followed by induction of a regulated state that correlated with failure of virus to induce T1D in older rats. In contrast, the BBDR.lyp/lyp temporal signature exhibited asynchronous dynamics, with delayed induction of inflammatory transcription and later, weaker induction of regulatory transcription, consistent with their deficiency in regulatory T cells. Through longitudinal analyses of plasma-induced signatures in BB rats and a human T1D progressor, we have identified changes in immunoregulatory processes that attenuate a preexisting innate inflammatory state in BBDR rats, suggesting a mechanism underlying the decline in T1D susceptibility with age.

Depletion of RT6.1+ T lymphocytes induces diabetes in resistant biobreeding/Worcester (BB/W) rats.

To investigate the role of RT6+ T cells in the pathogenesis of diabetes in BB/W rats, we treated animals from the diabetes-resistant (DR) subline with anti-RT6.1 lymphocytotoxic mAb. This depleted greater than 95% of peripheral RT6+ T cells but did not substantially reduce levels of circulating T cells or the in vitro response of spleen cells to mitogen. Treatment of 30-d-old DR BB/W rats in this way: induced insulitis and diabetes, rendered nondiabetic RT6-depleted DR rats susceptible to the adoptive transfer of diabetes by spleen cells from acutely diabetic BB/W rats, and yielded DR spleen cell populations capable of the adoptive transfer of diabetes to diabetes-prone (DP) or DR recipients. Treatment of DR rats beginning at 60 d of age failed to produce these effects. These results suggest that both susceptibility and resistance to diabetes in the BB/W rat are in part regulated by the RT6+ T cell subset and provide evidence for the importance of regulatory T lymphocytes in the pathogenesis of autoimmunity and diabetes in BB/W rats.

Prevention of diabetes in the BB rat by essential fatty acid deficiency. Relationship between physiological and biochemical changes.

Essential fatty acid (EFA) deficiency exerts a striking protective effect in several animal models of autoimmune disease. We now report that EFA deprivation prevents diabetes in the BB rat, an animal model of human insulin-dependent diabetes mellitus. In diabetes-prone (DP)-BB rats, the incidences of spontaneous diabetes and insulitis (the pathological substrate of autoimmune diabetes) were greatly reduced by EFA deficiency. This beneficial effect of the deficiency state was also seen in diabetes-resistant (DR)-BB rats that, after treatment with antibody to eliminate RT6+ T cells, would otherwise have become diabetic. The susceptibility of EFA-deprived DP-BB rats to spontaneous diabetes was restored when they were given dietary supplements of linoleate at 70 d of age (during the usual period of susceptibility), but not when they were repleted beginning at 120 d (after the peak incidence of diabetes). EFA deficiency did lead to growth retardation, but calorically restricted control rats demonstrated that the protective effect of the deficiency state was not a function of decreased weight. To examine the relationship between the biochemical changes of EFA deficiency and its physiological effects in this system, we compared the fatty acid changes that occurred in EFA-deficient animals that did and did not develop diabetes. Nondiabetic animals had significantly lower levels of (n-6) fatty acids (i.e., linoleate and arachidonate) and higher levels of oleate, an (n-9) fatty acid, than did diabetic animals. Levels of 20:3(n-9), the fatty acid that uniquely characterizes EFA deficiency, were similar in both groups, however. Among diabetic EFA-deficient rats, the age at onset of diabetes was found to correlate inversely with the level of (n-6) fatty acids, the least depleted animals becoming diabetic earliest, whereas there was no correlation with levels of 20:3(n-9). Among animals repleted with linoleate beginning at 70 d, restoration of susceptibility to diabetes correlated with normalization of the level of arachidonate. In summary, EFA deprivation reduced the frequency of diabetes in both DP and RT6-depleted DR-BB rats. This protective effect was strongly associated with depletion of (n-6) fatty acids, particularly arachidonate, but not with accumulation of the abnormal 20:3(n-9). Conjecturally, arachidonate and/or a metabolite may play a key role in mediating inflammatory injury in this animal model of autoimmune diabetes.

Genetic dissection reveals diabetes loci proximal to the gimap5 lymphopenia gene.

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

Tumor-induced anorexia in the Wistar rat.

The transplantable Leydig cell tumor of Wistar rats, LTW(m), caused decreased food consumption and weight loss in the host within 2 weeks of implantation. The tumor was small, did not metastasize, and did not affect several parameters of biochemical function. When the tumors were removed, increases in food intake and body weight occurred within 72 hours and were sustained. Reimplantation of tumors caused anorexia to recur. Parabiotic pairs of rats with tumor in one partner also experienced weight loss. Those rats in parabiosis with tumor-bearing rats gained less weight than those in parabiosis with control rats. These observations suggest that the LTW(m) tumor causes anorexia and that this anorexia is mediated by a circulating substance.

Transfusions of whole blood prevent spontaneous diabetes mellitus in the BB/W rat.

Weekly transfusions of whole blood from a nondiabetic subline of BB/W rats reduced the incidence of diabetes in susceptible BB/W rats from 39 to 0 percent and the incidence of pancreatic insulitis from 64 to 6 percent. Responsiveness of lymphocytes to concanavalin A was found to be low in rats with diabetes or insulitis. Transfusion restored concanavalin A responsiveness to levels observed in control rats free of diabetes or insulitis. These data suggest that whole blood alters the course of autoimmune BB/W rat diabetes.

Recapitulation of normal and abnormal BB rat immune system development in scid mouse/rat lymphohemopoietic chimeras.

Mice homozygous for the mutation "severe combined immune deficiency" (C.B17-scid/scid) lack functional T and B lymphocytes and readily accept tumor xenografts. Partial lymphohemopoietic scid/human and mouse/rat chimeras have been described, but complete chimerization with thymic engraftment and generation of donor-origin thymocytes has not been achieved. We now report that low-dose irradiation permits the engraftment of BB rat fetal liver stem cells in scid recipients. We observed that BB rat fetal liver cells injected into irradiated scid mice establish a rat hemopoietic system in the scid mouse bone marrow and populate the scid mouse thymus. These stem cells generated rat-origin thymocytes that migrated to the scid mouse spleen, a peripheral lymphoid organ. Finally, we found that xenogeneic chimeras created using fetal liver cells from the abnormal (lymphopenic, diabetes prone) subline of BB rats recapitulated both the quantitative and phenotypic abnormalities of the donor rat. Xenogeneic lymphohemopoietic chimeras established in scid mice may provide a powerful new tool in the study of immune system development and autoimmunity.

Spleen cell transfusion in the Bio-Breeding/Worcester rat. Prevention of diabetes, major histocompatibility complex restriction, and long-term persistence of transfused cells.

We report that transfusions of RT1u Wistar-Furth (WF) spleen cells prevented spontaneous diabetes in the RT1u BB/W rat while RT1b Buffalo rat spleen cells did not. In addition, donor origin WF T lymphocytes were detected in nondiabetic-susceptible BB/W recipients 5 mo after transfusion. Survival of donor-origin lymphocytes may provide the cellular mechanism by which major histocompatibility complex-compatible WF spleen cell transfusions prevent BB rat diabetes.

Lymphocyte transfusions prevent diabetes in the Bio-Breeding/Worcester rat.

The Bio-Breeding/Worcester (BB/W) rat develops spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. Transfusions of whole blood from the nondiabetic W-line of BB/W rats prevent the syndrome in diabetes-prone recipients. We report three experiments designed to determine which blood component is protective. In all experiments, diabetes-prone BB/W rats 23 to 35 d of age were given four or six weekly intravenous injections. In the first experiment, animals received either saline or transfusions of erythrocytes, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/22 (32%) erythrocyte, 2/27 (7%) white cell, 14/24 (58%) plasma, and 15/27 (56%) saline recipients (P less than 0.001). At 120 d of age, peripheral blood was obtained from nondiabetic rats. Fluorescence-activated cell sorter analysis of OX 19 tagged leucocytes revealed 35% T lymphocytes in white cell recipients (n = 13), compared with 9% in saline recipients (n = 7; P less than 0.001). Responsiveness to concanavalin A was also increased in the white cell group, whereas the frequency of both insulitis and thyroiditis was decreased. In the second experiment, 1/19 (5%) rats transfused with W-line spleen cells developed diabetes, as contrasted with 12/18 (67%) recipients of diabetes-prone spleen cells and 19/31 (61%) noninjected controls (P less than 0.001). In the third experiment, diabetes-prone rats received either W-line blood treated with a cytotoxic anti-T lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20 (40%), 1/20 (5%), and 13/19 (68%) rats in each group, respectively (P less than 0.001). We conclude that transfusions of W-line T lymphocytes prevent diabetes in the BB/W rat.

Diabetes in the Bio-Breeding/Worcester rat. Induction and acceleration by spleen cell-conditioned media.

Injections of media conditioned by concanavalin A-activated spleen cells from acutely diabetic rats accelerated the appearance of diabetes in young Bio-Breeding/Worcester (BB/W) rats. Activity was also found in media conditioned by spleen cells from nondiabetic, W-line Wistar Furth and Buffalo rats. Unconditioned media containing mitogen had no activity. Conditioned media also induced diabetes in resistant W-line BB/W rats but not in Wistar Furth rats. A soluble factor may activate a BB lymphocyte population that promotes diabetes.

High stimulatory activity of dendritic cells from diabetes-prone BioBreeding/Worcester rats exposed to macrophage-derived factors.

Dendritic cells (DC) present antigen and initiate T cell-mediated immune responses. To investigate the possible association of autoimmunity with DC function, we compared the accessory activity of splenic DC from Wistar/Furth (WF) and diabetes-prone (DP) BioBreeding (BB) rats. The latter develop autoimmune diabetes and thyroiditis. DC function was quantified in vitro by measuring T cell proliferation in mitogen-stimulated and mixed lymphocyte reactions. When purified without macrophage coculture, WF and DP DC displayed similar levels of accessory activity. In contrast, when purified by a method involving coculture with macrophages, DC from DP rats consistently displayed greater accessory activity. This finding could not be explained by morphological or phenotypic differences between DP and WF DC. In accessory activity assays performed after reciprocal DC cocultures with DP and WF macrophages, DP DC exhibited higher accessory activity irrespective of macrophage donor strain. We also compared the accessory activity of WF and DP DC cultured in the presence of conditioned medium and a mixture of IL-1 and GM-CSF. In all assays, DP DC exhibited higher accessory activity. In studies of (WF x DP) F1 hybrids, the high accessory activity of DP DC was observed to be heritable, and studies of WF and DP radiation chimeras indicated that the effect was an intrinsic property of the DP hematopoietic system. We conclude: (a) splenic DC from DP and WF rats possess similar basal levels of accessory potency; (b) after interaction with macrophages, DC of DP origin are capable of greater stimulatory activity than are WF DC; and (c) the mechanism responsible for this phenomenon involves differential responsiveness of DP and WF DC to macrophage-derived factors such as IL-1 and GM-CSF.

Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4(+) T cells, interferon-gamma, and CTLA4.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.

The pathogenesis of autoimmune diabetes mellitus.

There is increasing evidence that both DP and DR BB rats fail to clonally delete autoreactive T cells in the thymus that are important in the development of autoimmune IDDM. The DP BB rat also has a defect in its ability to generate a regulatory (RT6+) T-cell population that would prevent the onset of diabetes and, therefore, it becomes spontaneously diabetic. The DR rat develops autoreactive T cells, but does not express diabetes because of the concurrent development of a regulatory (RT6+) T-cell population. We suggest that in the BB rat, the initial immunological lesion is orchestrated by an APC in close proximity to pancreatic islet beta cells, and may be specifically directed to the beta cell itself. The release of cytokines in the vicinity of the beta cell destroys this highly susceptible target, causing the release of beta cell 'autoantigens'. These autoantigens, in turn, target autoreactive T cells to the beta cells, allowing a focal destructive process to spread throughout the pancreas. The ultimate destruction of the islets and the development of diabetes result from a cascading effect of this process, with the recruitment of other non-specific immune mediators. A similar process may also be initiated by APC within the thyroid of the rat, resulting in thyroiditis. The fact that the thyrocyte does not die is unexplained, but it could relate to the relative insensitivity of this cell type to various cytokines.

Anti-endothelial cell autoantibodies in BB rats with spontaneous and induced IDDM.

Anti-endothelial cell (anti-EC) antibodies occur in several autoimmune diseases, including human IDDM, but the time course of their development and their importance in disease pathogenesis are unknown. To study such antibodies further, we investigated the BB rat model of autoimmunity. Diabetes-prone (DP) BB rats spontaneously develop autoimmune diabetes, whereas coisogenic diabetes-resistant (DR) BB rats are disease free but can be induced to become diabetic by the depletion of T-cells expressing the RT6 alloantigen. Anti-EC autoantibodies were readily detectable in both untreated DP-BB rats and RT6-depleted DR-BB rats before the onset of diabetes. Their concentration increased with time. The anti-EC antibodies in DP-BB rats were almost exclusively of the IgG2b subclass, whereas those in RT6-depleted DR-BB rats included both the IgG1/2a and the IgG2b subclasses. We also found that intravenous injections of purified immunoglobulins from RT6-depleted DR-BB rats induced abnormal pancreatic vascular leakage in mice. The preabsorption of immunoglobulins against cultured ECs abolished this activity. The pretreatment of mice with silica also abolished the ability of immunoglobulins of RT6-depleted DR-BB rats to induce pancreatic leakage, suggesting that monocytes are involved in the mechanism of anti-EC autoantibody-induced vascular leakage. We conclude that anti-EC autoantibodies are present in rat strains that are genetically predisposed to develop autoimmune diabetes. Their presence early in the disease process and their ability to induce pancreatic vascular leakage suggest that they may participate in diabetes pathogenesis.

DR-BB rat thymus contains thymocyte populations predisposed to autoreactivity.

We have induced autoimmune insulin-dependent diabetes mellitus (IDDM) in athymic WAG rats by transfusing thymocytes from histocompatible phenotypically normal rats of the DR-BB strain. DR-BB rats rarely develop spontaneous IDDM, but readily become hyperglycemic if depleted in vivo of regulatory T-cells that express the RT6.1 maturational alloantigen. Successful adoptive transfer of IDDM by DR-BB thymocytes required that the athymic recipients be depleted of emerging populations of donor-origin RT6.1+ T-cells. Thymocytes from both normal and RT6-depleted diabetic DR donors were equally capable of transferring autoimmunity. In contrast, thymocytes from normal histocompatible YOS rats failed to transfer IDDM. The autoreactive potential of DR-BB rat thymocytes was minimal from birth to 4 weeks of age and then increased substantially at 8-9 weeks of age. These results demonstrate that the DR-BB rat thymus harbors abnormal cell populations predisposed to autoreactivity. The data localize the developmental defect leading to diabetes in the BB rat to an abnormal intrathymic selection process.

Speculations on etiology of diabetes mellitus. Tumbler hypothesis.

Although clinically useful, the conventional partition of diabetes mellitus into two major classes, insulin dependent and non-insulin dependent, has become obsolete from an etiological standpoint. Contemporary research, particularly that with advanced cellular and molecular methodologies, suggests that the expression of diabetes depends on a wide range of factors. We suggest that the etiology of diabetes has become analogous to the cylinder of a lock containing many tumblers. Each tumbler, e.g., environment, genetics, or cellular interactions, must be aligned before the key can be turned and an understanding of the etiologic process claimed.

Role of host immune system in BB/Wor rat. Predisposition to diabetes resides in bone marrow.

The role of the immune system in the development of autoimmune diabetes mellitus in the BB/Wor rat was studied with bone marrow transplantation methodology. In the first experiment, diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats were irradiated and reconstituted with bone marrow to create both reciprocal (DP donor----DR host; DR donor----DP host) and syngeneic (DR----DR; DP----DP) histocompatible chimeras. Both susceptibility and resistance to subsequent spontaneous diabetes in these chimeras were found to be a function of the type of donor bone marrow transplanted and not the genetic background of the host. In a second experiment, rats from three strains that share the RT1u major histocompatibility complex haplotype of the BB/Wor and rats from three non-RT1u strains were lethally irradiated and reconstituted with DP BB/Wor bone marrow. To rapidly induce diabetes and/or insulitis, they were then injected with mitogen-activated spleen cells from acutely diabetic DP BB/Wor donors, with standard passive-transfer methods. Diabetes and pancreatic insulitis were observed in RT1u recipients, whereas non-RT1u rats developed insulitis but not diabetes. The data suggest that predisposition to spontaneous diabetes in BB rats resides in bone marrow cells.

Dietary cow's milk protein does not alter the frequency of diabetes in the BB rat.

One theory of the pathogenesis of IDDM proposes that exposure to cow's milk proteins triggers the disease in genetically susceptible individuals. We tested this hypothesis in the BB/Wor rat model of human IDDM. Diabetes-prone (DP) BB/Wor rats spontaneously develop IDDM. Coisogenic diabetes-resistant (DR) BB/Wor rats do not develop diabetes spontaneously, but IDDM can readily be induced by treatment with polyinosinic:polycytidylic acid and depletion of RT6+ T-cells. Pregnant BB/Wor rats were fed one of four experimental diets or a standard Purina commercial rat chow (5010) that was certified to be free of cow's milk protein. Offspring were maintained on the maternal diet after weaning. DP-BB/Wor rats, fed either of two experimental diets based on hydrolyzed casein and free of intact milk protein (Nutramigen or D11236), developed diabetes at only half the rate of animals fed Purina 5010 chow. Neither the addition of bovine serum albumin (BSA) to Nutramigen nor the substitution of total milk protein for the hydrolyzed casein in the D11236 diet increased the frequency of spontaneous diabetes. In contrast, there was no relationship between diet and susceptibility of DR-BB/Wor rats to IDDM induction. However, the methods used to induce IDDM in DR-BB/Wor animals were found to induce antibodies against BSA. We conclude the following: 1) Dietary modification can reduce spontaneous IDDM expression in DP-BB/Wor rats, but the agent of protection is not elimination of cow's milk protein. 2) The addition of BSA or intact milk protein does not abrogate the effectiveness of a protective diet. 3) The genetic susceptibility of the DR-BB/Wor rat to autoimmune diabetes is unaffected by any of the tested diets, but a role of anti-BSA-like autoreactivity in IDDM expression cannot be excluded.

Spontaneous diabetes in the gnotobiotic BB/W rat.

To determine the influence of infectious agents on the initiation of diabetes in the spontaneously diabetic Bio-Breeding/Worcester (BB/W) rat, susceptible rats were raised in a germ-free environment. Between 2 and 3 mo of age, 3 of 12 pups became diabetic. Histologic examination of the pancreas revealed insulitis or end-stage islets. Culture and smears from various tissues were negative for bacteria or parasites. Serum vital antibody titers for all known rat viruses were undetectable. These data suggest that the diabetic syndrome of the BB/W rat is not dependent on recognized infectious agents.