Helle Markholst

Ian4 is required for mitochondrial integrity and T cell survival

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.

Neonatal Tolerization With Glutamic Acid Decarboxylase But Not With Bovine Serum Albumin Delays the Onset of Diabetes in NOD Mice

To test the role of glutamic acid decarboxylase (GAD65) or bovine serum albumin (BSA) autoimmunity in the pathogenesis of diabetes, GAD65 or BSA was injected intraperitoneally into neonatal female NOD mice (100 micrograms/mouse of each protein). Treatment with GAD65, but not with BSA, significantly delayed the onset of diabetes compared with control mice (P < 0.05). At 18 weeks, 6 of 10 control mice compared with 0 of 10 GAD65-treated mice (P = 0.005) and 7 of 14 BSA-treated mice had developed diabetes. However, after 79 weeks, 6 of 10 of the GAD65-treated mice were diabetic compared with 9 of 10 of the control mice and 12 of 14 of the BSA-treated mice. In GAD65-treated mice without diabetes, insulitis was markedly reduced compared with control or BSA-treated mice (P < 10−4). To further elucidate why GAD becomes an autoantigen, the expression in NOD mice islets was studied. Quantitative immunohistochemistry revealed that islet cell expression of GAD was increased in 5-week-old NOD mice compared with BALB/c mice (P = 0.02). With the occurrence of insulitis (9–15 weeks), the GAD expression was further increased relative to 5-week-old NOD mice (P < 0.02). In conclusion, GAD, but not BSA, autoimmunity is important for the development of diabetes in NOD mice. Furthermore, concordant with the appearance of insulitis, the GAD expression increased in NOD mouse islets, which could possibly potentiate the β-cell-directed autoimmunity.

Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice.

A role for the activating NK‐receptor NKG2D has been indicated in several autoimmune diseases in humans and in animal models of type 1 diabetes and multiple sclerosis, and treatment with monoclonal antibodies to NKG2D attenuated disease severity in these models. In an adoptive transfer‐induced model of colitis, we found a significantly higher frequency of CD4+NKG2D+ cells in blood, mesenteric lymph nodes, colon, and spleen from colitic mice compared to BALB/c donor‐mice. We, therefore, wanted to study the effect of anti‐NKG2D antibody (CX5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. CX5 treatment decreased the detectable levels of cell‐surface NKG2D and prophylactic administration of CX5 attenuated the development of colitis significantly, whereas a more moderate reduction in the severity of disease was observed after CX5 administration to mildly colitic animals. CX5 did not attenuate severe colitis. We conclude that the frequency of CD4+NKG2D+ cells increase during development of experimental colitis. NKG2D may play a role in the early stages of colitis in this model, since early administration of CX5 attenuated disease severity.

Characterisation of enterocolitis in the piroxicam-accelerated interleukin-10 knock out mouse — A model mimicking inflammatory bowel disease

BACKGROUND In inflammatory bowel disease a defective mucosal barrier, a dysregulated immune response and an excessive reactivity against the gut microbiota are assumed to cause a breakdown of the intestinal homeostasis and lead to chronic inflammation. Piroxicam treatment is a method for induction of colitis in IL-10 k.o. mice, which integrates a dysfunction of both the intestinal barrier and the immune system. However, the translational value of this model has not been thoroughly clarified. AIM To characterise the piroxicam-accelerated colitis (PAC) IL-10 k.o. model with respect to clinical features, pathogenic mechanisms and its ability to respond to existing therapies. METHODS The PAC IL-10k.o. model was established on a C57BL/6J background and the clinical manifestations, immunological mechanisms and efficacy of ampicillin and anti-IL-12/23p40 treatment were assessed. RESULTS The PAC IL-10 k.o. mice developed weight loss and diarrhoea, and colonoscopy revealed a thickened granulomatous mucosa. Histological evaluation of ileum and colon showed Crohns disease-like changes with pronounced hyperplasia and focal transmural inflammation. Ileitis was also observed in piroxicam treated wild type mice. The total number of neutrophils, monocytes and natural killer cells was elevated in the blood compared to IL-10 k.o. and wild type mice, indicating a role of the innate immune system in the pathogenesis. These findings were supported by analyses of the intestinal cytokine profile. Ampicillin and anti-IL-12/23p40 treatment significantly suppressed disease in the model. CONCLUSION The PAC IL-10 k.o. model resembles several features of Crohns disease and could be a useful in vivo model in preclinical research.

Pharmacological Evaluation of the SCID T Cell Transfer Model of Colitis: As a Model of Crohn's Disease

Animal models are important tools in the development of new drug candidates against the inflammatory bowel diseases (IBDs) Crohns disease and ulcerative colitis. In order to increase the translational value of these models, it is important to increase knowledge relating to standard drugs. Using the SCID adoptive transfer colitis model, we have evaluated the effect of currently used IBD drugs and IBD drug candidates, that is, anti-TNF-α, TNFR-Fc, anti-IL-12p40, anti-IL-6, CTLA4-Ig, anti-α4β7 integrin, enrofloxacin/metronidazole, and cyclosporine. We found that anti-TNF-α, antibiotics, anti-IL-12p40, anti-α4β7 integrin, CTLA4-Ig, and anti-IL-6 effectively prevented onset of colitis, whereas TNFR-Fc and cyclosporine did not. In intervention studies, antibiotics, anti-IL-12p40, and CTLA4-Ig induced remission, whereas the other compounds did not. The data suggest that the adoptive transfer model and the inflammatory bowel diseases have some main inflammatory pathways in common. The finding that some well-established IBD therapeutics do not have any effect in the model highlights important differences between the experimental model and the human disease.

Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice

IntroductionRheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.MethodsDTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.ResultsWe demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.ConclusionsOur findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.

An F344 rat congenic for BB/DP rat-derived diabetes susceptibility loci Iddm1 and Iddm2

The diabetes-prone BB rat (BB/DP) spontaneously develops an insulin-dependent diabetes resembling human Type 1 diabetes (previously named IDDM). As in Type 1 diabetes, the insulinproducing b-cells of the islets of Langerhans are targets for autoimmune destruction (Crisa et al. 1992). Two major diabetes susceptibility genes have been genetically positioned from this model: Iddm1 (Lyp) on rat chromosome (Chr) 4 and Iddm2 (RT1) on rat Chr 20 (Colle et al. 1981; Jacob et al. 1992), but several studies have defined additional loci involved in diabetes development (Kloting et al. 1995, 1998; Kloting and Kovacs 1998; Martin et al. 1999; Klaff et al. 1999). In the latter reference, a locus on Chr 2 (Iddm3), where the F344 allele protects against diabetes, was detected. Because the F344 rat also carries protective alleles of Iddm1 and -2, a precise positioning of this locus was not performed. We set out to produce an F344 rat congenic for the Iddm1 and -2 of BB/DP rat origin (F344.lypRT1). Such a strain will make precise positioning of Iddm3 (and other genes affecting diabetes) more feasible, since fewer animals need to be bred. The F344.LypRT1 strain originated in F2(F344 × BB/DP) rats that were backcrossed to F344. These animals were then intercrossed, and offspring were selected for homozygocity for the BB/DP alleles of Lyp and RT1 by FACS (described in Jackerott et al. 1997). At this point, marker-assisted selection was started (Wakeland et al. 1997) in addition to monitoring the two Iddmloci. Four rounds of backcross to F344 followed, with selection of heterozygotes for RT1 by FACS and for Lyp by PCR [via flanking markers D4Rat75 and D4Mit7 (Npy) (described in Hornum and Markholst 1999)]. These rats were designated “N6”F344.LypRT1, since six rounds of introgression had been performed. For marker-assisted selection, 86 microsatellites evenly distributed throughout the genome were typed in each generation, and animals with the highest number of markers of F344 origin were selected for further breeding. The longest distance between markers (or markers and chromosome ends) is 46 cM. “N6”F344.LypRT1 rats, with all 86 microsatellites of F344 origin, were finally intercrossed, and offspring homozygous for the BB/ DP-derived Iddm1and Iddm2-alleles form the basis of the new F344.LypRT1 congenic strain. The segment of BB/DP origin around Iddm1 on Chr 4 is delimited by D4Rat15 and D4Rat37 with map positions 29.4 cM and 46.5 cM on the SHRSP × BN rat genetic map from the Whitehead Institute (www.genome.wi.mit.edu/rat/public), making the maximal size of the BB/DP-derived segment 17.1 cM. The segment of BB/DP origin around Iddm2 on Chr 20 is delimited by the centromere end and D20Rat43 and -70, both of which map to position 15.7 cM on the FHH × ACI rat genetic map from the Whitehead Institute, making the maximal size of the BB/DP derived segment 15.7 cM. While the diabetes incidence in our BB/DP line is >90% with more than 99% of these becoming diabetic before the age of 120 days, none of 22 (0%) F344.LypRT1 congenic rats became diabetic at this age (p << 0.001), although kept under the same specific pathogen-free conditions. Insulitis was not detected among four F344.LypRT1 congenic rats at the age of 120 days. This clearly demonstrates that loci outside Iddm1 and -2 protect

Lack of Systematically Found Insulin Autoantibodies in Spontaneously Diabetic BB Rats

Insulin autoantibodies (IAAs) occur in newly diagnosed human insulin-dependent diabetes mellitus (IDDM) patients, but their presence in BB rats is controversial, possibly due to assay differences or variability in the animals studied. To resolve this controversy, lAAs were measured in well-characterized inbred BB rats both in radioligand assays with 125I-labeled rat insulin I or II, respectively, and in an enzyme-linked immunosorbent assay (ELISA) with rat insulin as antigen. In prospective studies, a total of 57 serums from 16 diabetes-prone (DP) BB rats were obtained during an interval ranging from 15 wk to the last week before onset and at onset of diabetes. At comparable ages, 21 serums were obtained from 8 DP BB rats not developing diabetes, and 70 matched serums were obtained from 19 diabetes-resistant (DR) BB rats. Levels of antibody binding increased slightly with increasing age in DP and matched DR rats. Two rats were positive at onset of IDDM in all assays but not in earlier samples. Otherwise, only few isolated serums from both types of rats regardless of diabetes had increased binding in one of the assays. In a cross-sectional study, the insulin-binding levels in 150-day-old DP rats (n = 20) that had not yet developed diabetes did not correlate with insulitis present in 3 of 20 rats and did not differ from 150-day-old DR BB rats (n = 20). Diabetic BB rats (n = 20) killed at 150 days of age after treatment with protamine zinc insulin for 1–3 mo showed increased binding levels (P < 0.01–0.001) in all assays compared with the DR and DP rats. We conclude that 1) IAAs may be present at low frequency at onset of IDDM in BB rats, 2) different patterns of reactivity are found in the two radioligand assays and in the ELISA, and 3)IAAs are not a marker for later development of IDDM or insulitis in BB rats.

CD40 Is Required for Development of Islet Inflammation in the RIP-CD154 Transgenic Mouse Model of Type 1 Diabetes

Abstract:  Type 1 diabetes is believed to be an autoimmune disease where cells of the immune system destroy the insulin‐producing β cells in the islets of Langerhans. The trigger(s) of the inflammatory reaction is yet unknown, but both genetic and environmental factors, including viruses or other pathogens, are thought to play a role. We have recently described a transgenic mouse model—the RIP‐CD154 mouse—in which β‐cell–specific expression of CD154 (CD40 ligand) mediates immune activation, insulitis, and diabetes on a non–diabetes‐prone background. By the use of bone marrow chimeric mice, we now demonstrate that a functional Cd40 gene is necessary for islet inflammation and we show that CD40 expression on bone marrow–derived cells is sufficient to trigger activation of the immune system and development of insulitis.

PolyI:C Induction of Diabetes Is Controlled by Iddm4 in Rats with a Full Regulatory T Cell Pool

Abstract:  The Iddm4 gene controls diabetes in rats depleted of regulatory T cells (Treg) and immune‐activated via treatment with the toll‐like receptor 3 (TLR‐3) ligand, polyI:C. Both diabetes‐resistant (BBDR) and diabetes‐prone (BBDP) BB rats carry dominant permissive alleles of Iddm4, while the recessive Wistar Furth (WF) rat allele is protective. Iddm4 is positioned close to Iddm2 on chromosome 4, but when we introgressed BBDP‐derived parts of this region—either containing both genes or Iddm2 alone—into the WF genome, none of these congenic strains developed spontaneous diabetes. Although both strains harbor two copies of the recessive Iddm2 allele of the BBDP rat, making these animals devoid of Treg cells, immune activation in itself via polyI:C treatment did not induce overt diabetes. Interestingly, TLR‐3 ligation without depletion of Tregs resulted in diabetes and insulitis development in nonlymphopenic F1‐offspring of mating the Iddm4+Iddm2 congenic strain to WF. This demonstrates that the diabetogenic allele of Iddm4 is able to confer diabetes susceptibility even in a nonlymphopenic host with a full Treg pool, and that homozygosity for Iddm2—although responsible for an almost total lack of Tregs—delays the disease process. Finally, we have confirmed the position of Iddm4 in truly congenic strains.