Despite major advances in characterising the disease pathogenesis of sarcoidosis, it is still unclear which immune pathway is the main contributor to disease activity. The most effective treatment, corticosteroids, has a high adverse effect burden so there is a need to define more specific therapeutic agents with less side effects. In addition, for some patients with progressive active disease, corticosteroids are often unhelpful; therefore better understanding of the mechanisms of disease is needed for development new drugs. This study examines the potential immune processes involved in disease activity in sarcoidosis using gene expression profiling of peripheral blood mononuclear cells (PBMCs) (n=29) and bronchoalveolar lavage cells (BALCs) (n=12). Patients with well-defined pulmonary sarcoidosis and secure tissue-supported diagnosis were recruited from the Oxford Sarcoidosis Service during a defined 2 year period. Patients were not on treatment at the point of sampling. A CTAS1 -validated chest radiograph-blood disease activity score comprising lymphocyte, ACE and IgG levels (SCAS, scores from 0 to 12 reflecting low to high activity) was used to measure activity at the point of sampling. Gene expression profiles were derived from PBMC and BALCs using the Illumina HT-12 v4 expression chip. All RNA had a RIN≥8. We found a significant positive correlation between the ‘immune response’ gene set and SCAS for BALCs, by GSEA and Metacore functional analyses. Within this gene set, a transcriptional signature related to monocyte activity and function was shown to be the most significant gene network with an unexpected downregulation of TGFb receptor signalling pathway in low activity BALCs. In PBMCs and BALCs, the two IFN-g-inducible, monocyte-produced genes CXCL-9 and CXCL-10 were the soluble factors that most correlated with increasing activity (r≥0.5 by Spearman Correlation). In an independent cohort, SCAS levels were examined against CD14hi classical monocyte levels (n=40, same inclusion criteria). This showed a marked correlation between monocyte frequency and level of activity as measured by SCAS (r=0.67; p<0.001; Spearman Rank correlation). These Results implicate monocytes as a major contributor to disease activity in sarcoidosis and propose monocyte pathways as potential specific targets for new therapeutics in sarcoidosis. Reference 1. Benamore R, Kendrick Y, et al. Thorax 2017.

Les variations communes de séquence d'ADN existantes chez l'Homme modifient l'expression des gènes et contribuent à la susceptibilité aux maladies complexes. Notre objectif est d'établir la relation entre le contrôle transcriptionel du tissu adipeux blanc et la susceptibilité à l'adiposité et au diabète mis en évidence chez le rat spontanément diabétique de la souche Goto-Kakizaki (GK). En utilisant des puces à ADN Illumina, nous avons analysé l'expression d'environ 20,000 gènes dans le tissu adipeux blanc d'une population F2 (n = 138) derivée du GK et du rat contrôle Brown-Norway (BN) et d'une lignée congénique portant sur un fond génétique BN, les allèles GK à un locus génetique (QTL) lié à l'adiposité. Les loci liés à l'expression des gènes (eQTL) ont été identifiés dans la population F2 en utilisant R/QTL, validés dans la lignée congénique par qRT-PCR, et analysés en relation avec la séquence génomique du GK produite au laboratoire. Nous avons identifié sur le génome entier, 585 eQTLs statistiquement significatifs (LOD > 9). La région 1q33 (8,3Mb), qui coségrège avec un QTL d'adiposité, contient 172 gènes candidats positionnels, parmi lesquels 44 correspondent à un eQTL. Nous avons validé l'expression différentielle de 27 de ces 44 gènes dans la lignée congénique, qui montrent pour 48 % d'entre eux un effet de régulation transcriptionnelle en cis. L'analyse de 20,000 polymorphismes SNPs présents dans la région 1q33 nous a permis d'éliminer les eQTLs faux positifs et d'entreprendre l'analyse fonctionnelle de polymorphismes localisés sur les gènes candidats positionnels du QTL lié à l'adiposité, parmi lesquels le facteur de transcription ASCL3 (LOD > 43). L'utilisation combinée de données du transcriptome et de séquençage nous a permis d'élucider le contrôle transcriptionnel du tissu adipeux blanc chez un modèle de diabète et d'identifier des gènes candidats fonctionnels et positionnels au locus1q33 associé à l'adiposité chez le rat GK. P

MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility.

Background: Liver is exposed to bacterial components related to intestinal permeability.NOD1, a bacterial receptor, appears to regulate the crosstalk between innate and adaptive immunity and the functions of polymorphonuclear neutrophils (PMN) in intestinal diseases.No data are available concerning the role of NOD1 in PMN related liver diseases.Aim: To explore the regulatory role of NOD1 in the PMN-induced liver injury.Methods: Livers taken from Nod1 +/ + and Nod1 -/ -mice challenged with CCl 4 were examined; main functions of PMN isolated from Nod1 +/ + and Nod1 -/ -mice were studied.Migration tests were performed in Boyden chambers using chemoattractants.Phagocytosis capacity was tested using the Phagotest ® ; we studied the main inflammatory pathways in PMN and CD11b expression on PMN surface by FACS analysis.Results: We observed that NOD1 was expressed in hepatocytes and PMN at mRNA and protein levels.In PMN-mediated injury models in mice, we showed that: After CCl 4 exposure, livers of Nod1 -/ -mice disclosed more than 50% lower PMN infiltration within necrotic areas compared to Nod1 +/ + .A lower PMN infiltration was also observed in Nod1 -/ -mice using a thioglycolate-mediated peritonitis model; The lack of difference in the liver expression of mRNA chemokines between Nod1 -/ -and control mice.There were no constitutional defects in blood formula or in granuloblasts isolated from bone marrow in Nod1 -/ -mice.Taken together these results suggested an impaired PMN recruitment.Then, in ex-vivo studies, we focused on PMN functions: PMN isolated from Nod1 -/ - mice displayed a 90% decrease in migration capacity compared to the Nod1 +/ + PMN; FK565, a potent ligand of NOD1, increased significantly human PMN migration; Phagocytosis capacity of Nod1 -/ -neutrophils was decreased by more than 50% compared to Nod1 +/ + .In terms of mechanistic insights: upon FK565 and fMLP stimulation, p38, JNK and NFkB activations were altered in Nod1 -/ -PMN; expression of CD11b on the Nod1 -/ -PMN surface was significantly decreased compared to Nod1 +/ + .Conclusions: NOD1 is involved in the ability of PMN to migrate in the liver.In terms of mechanisms, the defect of NOD1 leads to altered functional activities of PMN.NOD1 may be an interesting target to regulate PMN-related liver injury.

Aims/hypothesis Dyslipidaemia is a main component of the insulin resistance syndrome. The inbred Goto-Kakizaki (GK) rat is a model of spontaneous type 2 diabetes and insulin resistance, which has been used to identify diabetes-related susceptibility loci in genetic crosses. The objective of our study was to test the genetic control of lipid metabolism in the GK rat and investigate a possible relationship with known genetic loci regulating glucose homeostasis in this strain. Materials and methods Plasma concentration of triglycerides, phospholipids, total cholesterol, HDL, LDL and VLDL cholesterol were determined in a cohort of 151 hybrids of an F2 cross derived from GK and non-diabetic Brown Norway (BN) rats. Data from the genome-wide scan of the F2 hybrids were used to test for evidence of genetic linkage to the lipid quantitative traits. Results We identified statistically significant quantitative trait loci (QTLs) that control the level of plasma phospholipids and triglycerides (chromosome 1), LDL cholesterol (chromosome 3) and total and HDL cholesterol (chromosomes 1 and 5). These QTLs do not coincide with previously identified diabetes susceptibility loci in a similar cross. The significance of lipid QTLs mapped to chromosomes 1 and 5 is strongly influenced by sex. Conclusion/interpretation We established that several genetic loci control the quantitative variations of plasma lipid variables in a GKxBN cross. They appear to be distinct from known GK diabetes QTLs, indicating that lipid metabolism and traits directly relevant to glucose and insulin regulation are controlled by different gene variants in this strain combination.

Extracellular matrix molecules such as elastin and collagens provide mechanical support to the vessel wall. In addition to its structural role, elastin is a regulator that maintains homeostasis through biologic signaling. Genetically determined minor modifications in elastin and collagen in the aorta could influence the onset and evolution of arterial pathology, such as hypertension and its complications. We previously demonstrated that the inbred Brown Norway (BN) rat shows an aortic elastin deficit in both abdominal and thoracic segments, partly because of a decrease in tropoelastin synthesis when compared with the LOU rat, that elastin gene polymorphisms in these strains do not significantly account for. After a genome-wide search for quantitative trait loci (QTL) influencing the aortic elastin, collagen, and cell protein contents in an F2 population derived from BN and LOU rats, we identified on chromosomes 2 and 14, 3 QTL specifically controlling elastin levels, and a further highly significant QTL on chromosome 17 linked to the level of cell proteins. We also mapped 3 highly significant QTL linked to body weight (on chromosomes 1 and 3) and heart weight (on chromosome 1) in the cross. This study demonstrates the polygenic control of the content of key components of the arterial wall. Such information represents a first step in understanding possible mechanisms involved in dysregulation of these parameters in arterial pathology.

Genetic studies in human populations and rodent models have identified regions of human chromosome 1q21-25 and rat chromosome 2 showing evidence of significant and replicated linkage to diabetes-related phenotypes. To investigate the relationship between the human and rat diabetes loci, we fine mapped the rat locus Nidd/gk2 linked to hyperinsulinemia in an F2 cross derived from the diabetic (type 2) Goto-Kakizaki (GK) rat and the Brown Norway (BN) control rat, and carried out its genetic and pathophysiological characterization in BN.GK congenic strains. Evidence of glucose intolerance and enhanced insulin secretion in a congenic strain allowed us to localize the underlying diabetes gene(s) in a rat chromosomal interval of approximately 3-6 cM conserved with an 11-Mb region of human 1q21-23. Positional diabetes candidate genes were tested for transcriptional changes between congenics and controls and sequence variations in a panel of inbred rat strains. Congenic strains of the GK rats represent powerful novel models for accurately defining the pathophysiological impact of diabetes gene(s) at the locus Nidd/gk2 and improving functional annotations of diabetes candidates in human 1q21-23.

Inbred strains of the laboratory rat are widely used for identifying genetic regions involved in the control of complex quantitative phenotypes of biomedical importance. The draft genomic sequence of the rat now provides essential information for annotating rat quantitative trait locus (QTL) maps. Following the survey of unique rat microsatellite (11,585 including 1648 new markers) and EST (10,067) markers currently available, we have incorporated a selection of 7952 rat EST sequences in an improved version of the integrated linkage-radiation hybrid map of the rat containing 2058 microsatellite markers which provided over 10,000 potential anchor points between rat QTL and the genomic sequence of the rat. A total of 996 genetic positions were resolved (avg. spacing 1.77 cM) in a single large intercross and anchored in the rat genomic sequence (avg. spacing 1.62 Mb). Comparative genome maps between rat and mouse were constructed by successful computational alignment of 6108 mapped rat ESTs in the mouse genome. The integration of rat linkage maps in the draft genomic sequence of the rat and that of other species represents an essential step for translating rat QTL intervals into human chromosomal targets.

AIMS/HYPOTHESIS:Genetic investigations in the spontaneously diabetic (Type 2) Goto Kakizaki (GK) rat have identified quantitative trait loci (QTL) for diabetes-related phenotypes. The aims of this study were to refine the chromosomal mapping of a QTL ( Nidd/gk5) identified in chromosome 8 of the GK rat and to define a pathophysiological profile of GK gene variants underlying the QTL effects in congenics.METHODS:Genetic linkage analysis was carried out with chromosome 8 markers genotyped in a GKxBN F2 intercross previously used to map diabetes QTL. Two congenic strains were designed to contain GK haplotypes in the region of Nidd/gk5 transferred onto a Brown Norway (BN) genetic background, and a broad spectrum of diabetes phenotypes were characterised in the animals.RESULTS:Results from QTL mapping suggest that variations in glucose-stimulated insulin secretion in vivo, and in body weight are controlled by different chromosome 8 loci (LOD3.53; p=0.0004 and LOD4.19; p=0.00007, respectively). Extensive physiological screening in male and female congenics at 12 and 24 weeks revealed the existence of GK variants at the locus Nidd/gk5, independently responsible for significantly enhanced insulin secretion and increased levels of plasma triglycerides, phospholipids and HDL, LDL and total cholesterol. Sequence polymorphisms detected between the BN and GK strains in genes encoding ApoAI, AIV, CIII and Lipc do not account for these effects.CONCLUSIONS/INTERPRETATION:We refined the localisation of the QTL Nidd/gk5 and its pathophysiological characteristics in congenic strains derived for the locus. These congenic strains provide novel models for testing the contribution of a subset of GK alleles on diabetes phenotypes and for identifying diabetes susceptibility genes.