Lars Hornum

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.

Rapid‐onset clinical and mechanistic effects of anti‐C5aR treatment in the mouse collagen‐induced arthritis model

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti‐C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti‐murine C5aR (anti‐mC5aR) surrogate antibody in mouse collagen‐induced arthritis (CIA). First, efficacy was demonstrated in a multiple‐dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti‐mC5aR‐treated mice. Then, the mechanism of action was examined by looking for early effects of anti‐mC5aR treatment in single‐dose treatment studies. We found that 48 h after single‐dose treatment with anti‐mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose‐setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti‐C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

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

Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model.

Abstract Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.

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.

Comparative mapping of rat Iddm4 to segments on HSA7 and MMU6.

Iddm4 is one of several susceptibility genes that have been identified in the BB rat model of type 1 diabetes. The BB rat allele of this gene confers dominant predisposition to diabetes induction by immune perturbation in both the diabetes-prone and the diabetes-resistant substrains, whereas the Wistar Furth (WF) allele confers resistance. We have positioned the gene in a 2.8-cM region on rat Chromosome (Chr) 4, proximal to Lyp/Ian4l1. We have produced a radiation hybrid map of the Iddm4-region that includes a number of rat genes with their mouse and human orthologs. We present a comparative map of the rat Iddm4 region in rat, human, and mouse, assigning the gene to a 6.3-Mb segment between PTN and ZYX at 7q32 in the human genome, and to a 5.7-Mb segment between Ptn and Zyx in the mouse genome.

Mapping of caspase-2 in the rat and its exclusion as a candidate gene for lymphopenia.

Abstract. Caspase-2 is a member of the caspase family of cystein proteases involved in programmed cell death or apoptosis. Functional and genetic data suggest it as a candidate gene for lymphopenia (Lyp)—a susceptibility gene for rat diabetes—which is responsible for the T-cell lymphopenia in the diabetes–prone BB rat. Firstly, there is a higher frequency of apoptosis among recent thymic emigrants in the diabetes-prone BB rat than in the non-lymphopenic diabetes-resistant BB rat. Secondly, caspase-2 maps close to Tcrb on mouse Chromosome (Chr) 6, while Lyp is closely linked to Tcrb on the homologous rat Chr 4. In this paper, we report genetic fine-positioning and radiation hybrid mapping of caspase-2 in the rat. Both methods positioned caspase-2 to rat Chr 4 between markers Prss1 and D4Mit5. Since Lyp maps distally to D4Mit5, between markers D4Rat75 and Npy, we exclude caspase-2 as a candidate gene for Lyp.

The BB Rat

Diabetes-prone BioBreeding (BBDP) rats develop a spontaneous diabetic syndrome resembling human type 1 diabetes (T1D). Foremost, BBDP rats lose the majority of their beta cells during a rapid and aggressive inflammation of the islets during the last 2 weeks prior to overt hyperglycemia (1 ). Once hyperglycemia occurs, the rats become highly polyuric – they may lose well over 20 g (10–15% of body weight) overnight despite an excessive drinking behavior – and they proceed within 24–48 h to become ketotic. They die in ketoacidosis unless rescued by exogenous insulin therapy within the first 2 days of hyperglycemia (2, 3) – i.e. this clearly separates their diabetic phenotype from that of NOD mice that can easily survive for longer time after the onset of clinical diabetes. Indeed, within the first week, all insulin release and immunoreactivity in the pancreas is entirely lost – i.e. the phenotype of hypoinsulinemia is complete in BBDP rats (4–7).

AB1206 Dynamic contrast enhanced (DCE)-mri in relation to inflammatory markers in serum and joint fluid: initial data and validation in four most common knee arthritic diseases

Background Biomarker science has advanced to aid in distinguishing between different forms of arthritis: inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA) and osteoarthritis (OA). Biomarkers are also used to assess disease activity. Diagnostic serum biomarkers such as rheumatoid factor (RF) and cyclic-citrullinated peptide (CCP) and assays of disease activity such as C-reactive protein (CRP), and multi-biomarker assays have utility but lack complete sensitivity and specificity. Increasingly quantitative imaging biomarkers may fill an important gap in disease identification and assessment. Objectives 1) To investigate the association between imaging measures of inflammation in the synovium of the knee joint and systemic levels of CRP in patients with RA, PsA and OA. 2) Investigate how imaging and clinical markers correlate to IL-6 levels from joint fluid in different patient cohorts. Methods 38 patients with a flare of pain in the knee were recruited. 12 were diagnosed with RF positive (+) RA, 6 with RF negative (-) RA, 6 PsA, and 14 OA, according to ACR/EULAR criteria. CRP in blood and IL-6 levels from joint fluid were determined. Patients underwent MRI, including Dynamic Contrast Enhanced (DCE)-MRI exam prior to an ultrasound-guided arthrocentesis. MRI were scored for synovitis1 and DCE-MRI were quantified using Dynamic Enhanced MRI Quantification (DEMRIQ) method, extracting the volume of enhancing voxels (Nvoxel), Initial Rate of Enhancement (IRE), Maximum Enhancement (ME). Inflammation was quantified as IRExNvoxels and MExNvoxels.2 Correlation between all clinical scores and all imaging parameters was done using Spearman rho, with significance levels of p<0.05. Results The imaging markers of perfusion in the synovium of the knee (MExNvoxels and IRExNvoxels) were the only imaging measures, which showed a very high association with CRP in both RF +RA (r=0.92/0.97, p<0.05) and PsA patients (0.93/0.99, p<0.05), whereas all other imaging markers of inflammation showed no statistical association with blood levels of CRP in these diseases. We found no association between CRP and any imaging assessed scores of inflammation in either RF- RA or OA. In addition, only RF +RA patients showed a positive moderate to high association between MExNvoxels and IL-6 (r=0.66, p<0.05) in the knee joint aspirate. Conclusions Quantitative imaging and blood biomarkers of inflammation, such as DCE-MRI parameters and CRP, appear to relate differently to each other in the four most common knee arthritic diseases, RF +RA, RF- RA, PsA and OA. DCE-MRI may have specific utility in differentiating these conditions and their disease activity. References [1] Guermazi A, Roemer FW, Haugen IK, et al. MRI-based semiquantitative scoring of joint pathology in osteoarthritis. Nat Rev Rheumatol. 2013;9(4):236–5 [2] Kubassova Oet al; Eur J Radiol. 2010Jun;74(3):e67–72. Disclosure of Interest None declared

AB0079 Anti-c5a receptor antibody treatment ameliorates disease activity in delayed-type hypersensitivity (dth) arthritic mice

Background C5a is an anaphylatoxin generated in response to complement activation. C5a promotes leukocyte chemotaxis and activation via the C5a receptor (C5aR), both directly on C5aR-positive cells such as neutrophils, monocytes and macrophages, and indirectly on T cells via effects on C5aR-positive antigen specific cells (APCs)1. C5a production and C5aR signalling have been implicated in a wide range of inflammatory disorders including rheumatoid arthritis (RA)2. DTH arthritis is a single-paw arthritis model, which is dependent on CD4+ T cells. Besides T cells, neutrophils and macrophages are involved in the pathology, and inflammatory mediators associated with these cells are induced locally in the arthritic paw3 Objectives The purpose of this study was to investigate the therapeutic effect of a blocking anti-C5aR antibody in a single–paw arthritis model in mice in both a 2-week multiple-dose (MD) study and in a single-dose (SD) study with read-out at 60 h post-dosing. Methods DTH arthritis was induced as described by Atkinson3. In the MD study mice were treated twice weekly with anti-C5aR antibody (25 mg/kg/dose) from the day of arthritis induction. Mechanistic studies were conducted to investigate the immune-modulating effect of a SD of 25 mg/kg anti-C5aR with read-out at 60 h post-dosing/arthritis induction. Cytokine/chemokine levels in whole–paw homogenate supernatants were analyzed using ELISA and bioplex. Paw sections were evaluated histologically, and composition of leukocyte subsets isolated from lymph nodes and arthritic paws were analyzed by flow cytometry. Statistics: unpaired two-sided t-test. Results Significant reduction in paw swelling (P<0.01) and histopathology score (P<0.0001) was observed in the MDstudy. Swelling of the arthritic paw was also significantly reduced compared to control–treated mice in the SD study (P<0.0001). Also a single dose of anti-C5aR resulted in significantly reduced levels of MIP-2, MCP-1 and TNFα (P<0.05 for each analyte) in the arthritic paw compared to the levels found in control-treated mice. However, no difference was detected in the total number of leukocytes infiltrating the arthritic paws 60 h after SD treatment. Flow cytometric analysis of leukocyte subsets isolated from arthritic pawsafter SD treatment revealed significant reductions in CD4+ T cell counts of anti-C5aR-treated mice compared to control-treated mice (P<0.05), which correlated with significantly reduced CD4+ T cell proliferation (P<0.05) in the draining popliteal lymph node. Target validation in control-treated DTH arthritic mice revealed expression of C5aR on neutrophils (blood and paw) and on a subset of macrophages (paw), but not on CD4+ T cells. Conclusions Our studies reveal a significant effect of anti-C5aR treatment on DTH arthritis disease activity, and our mechanistic SD studies strongly suggests that the effect is mediated via reduced local production of inflammatory mediators and reduced CD4+ T cell proliferation, while no effect on leukocyte chemotaxis was observed 60 h after SD anti-C5aR antibody treatment. The effect on CD4+ T cell proliferation is probably indirect via effects of an anti-C5aR antibody on APCs. References Dunkelberger JR et al.(2010) Mol Immunol 47: 2176–2186; Lee H et al. (2008) Immunol Cell Biol 86: 153–160; Atkinson SA et al. (2012) Arthritis Res Ther 14(3):R134 Disclosure of Interest A. Nansen Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, S. Atkinson Employee of: PhD student, Research performed at Novo Nordisk A/S, P. Usher Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, C. Haase Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, L. Hornum Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S