The tristetraprolin family consists of mRNA destabilizing genes that bind to target mRNA and degrade them. In this context, TIS11 gene family is of primary interest in the spontaneous fetal loss seen in pigs. I hypothesized that the tristetraprolin family regulates these genes in this model. Higher levels of the TIS11 family transcripts in endometrium and trophoblast samples from healthy conceptuses at gd20 were found than in tissues from arresting conceptus attachment sites and significantly higher levels of TIS11 family transcripts were expressed in trophoblast samples associated with arresting conceptuses at gd50 compared to healthy endometrium and trophoblast. TIS11 proteins were expressed and localized at both maternal and fetal sides of maternal-fetal interface. These results provide a basis for understanding some aspects of gene regulation by mRNA destabilizing factors such as tristetraprolin at the maternal-fetal interface and how these factors may play a role in porcine pregnancy and fetal loss. / Department of Biomedical Sciences, NSERC, Ontario Pork, Bioniche Life Sciences Inc.

The NF-κB transcription factor is expressed in the majority of mammalian cells and regulates a large number of genes with important functions in a variety of cellular processes including cell growth, division, apoptosis and inflammatory responses. Perturbation of NF-κB response has been implicated in a variety of diseases such as asthma and inflammatory bowel disease, in addition to various forms of cancer. Through experiments at the single cell level it has been shown that NF-κB displays complex temporal activation, notably including nucleo-cytoplasmic oscillations. It has been observed that these oscillations occur in a heterogeneous manner; as such they are masked when measured at the population level. In contrast, pulsed TNFα treatment at 100 min intervals produces regular and synchronous nuclear peaks of NF-κB. Such pulsatile stimulation may reflect more accurately physiological conditions. The work in this project uses a Systems Biology approach consisting of bioinformatic, mathematical, and experimental methodologies to investigate how NF-κB can regulate such a diverse set of gene responses. Previously published studies have proposed that target gene expression levels following NF-κB activation (continuous TNFα) can be explained by a combination of key parameters, including transcript degradation rate, transcript structure, and transcription initiation rate. Initial work in this project highlighted that these explanatory factors are not sufficient to describe the observed temporal order of gene transcription. The roles of miRNAs and NF-κB subunit phosphorylation in regulation were additionally explored. A large set of genes was identified that are activated more strongly by pulsed TNFα than by continuous TNFα treatment. This suggests a new unreported mechanism of gene regulation, the possible causes of which are examined in this thesis. The gene list was refined by altering pulse frequency, which revealed an enrichment of NF-κB targets correlated with the regularity of these pulses. Temperature shift and anti-inflammatory drug treatment (Diclofenac) were shown to have a profound effect on NF-κB oscillation frequency. These perturbations provide an alternative method to study the effects of NF-κB oscillation frequency on specific target genes, independent of a pulse regime. Integration and analysis of these datasets suggested that a core, frequency-encoded set of genes regulated by NF-κB might exist. It is proposed that such genes may respond optimally to specific frequencies of NF-κB activation, implying a potential frequency threshold. The presence of such genes may explain the need for the complex systems that control NF-κB timing. It was noted that there was an enrichment of genes encoding transcription factors within the frequency encoding set, in addition to proteins which are known to be involved in the control of inflammation.

La retinosis pigmentaria (RP) es una forma de degeneración retiniana hereditaria caracterizada por la pérdida de los fotorreceptores (bastones y conos) de la retina. Constituye la principal causa genética de ceguera en los países desarrollados. Debido a su elevada heterogeneidad genética es difícil comprender los mecanismos patogenéticos responsables de la muerte de los fotorreceptores. Las mutaciones genéticas son las responsables de la apoptosis de los bastones. Sin embargo, la muerte de los conos parece debida a los cambios metabólicos que provoca la degeneración de los bastones como la hiperoxia (estrés oxidativo y nitrosativo), la secreción de diversos factores (citoquinas, quimioquinas) por parte de los bastones y otras células del entorno, etc. La muerte de los conos provoca la pérdida de la visión central en RP. El objetivo principal de este proyecto es evaluar el papel de la inflamación en pacientes con retinosis pigmentaria y en un modelo experimental, el ratón rd10. En concreto se determinará la presencia de factores inflamatorios en pacientes con RP y el efecto de la inhibición de la citoquina proinflamatoria, TNF¿, sobre la progresión de la enfermedad en el ratón rd10 y un modelo porcino ex vivo de degeneración retiniana. La caracterización y validación de este modelo ex vivo podría ser una alternativa útil para la comprensión de los mecanismos moleculares implicados en la muerte de los fotorreceptores en enfermedades retinianas. La inhibición del TNF¿ con anticuerpos específicos podría reducir el proceso inflamatorio y con ello, la muerte de los fotorreceptores en ratones rd10. Esto supondría el establecimiento de una nueva diana terapéutica para el desarrollo de tratamientos que previniesen o retrasasen el avance de la enfermedad en humanos. / Martínez Fernández De La Cámara, CI. (2015). Papel del estrés oxidativo y la inflamación en la retinosis pigmentaria. Efecto de la inhibición del TNFA en la progresión de la degeneración retiniana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48803 / TESIS

Objective: To test the hypothesis that loss of IL-19 exacerbates atherosclerosis. Approach and Results: Il19-/- mice were crossed into Ldlr-/- mice. Double knockout (dKO) mice had increased plaque burden in aortic arch and root compared to Ldlr-/- controls after 14 weeks of high fat diet (HFD). In a rescue study, dKO mice injected i.p. with 10ng/g/day of IL-19 had significantly less plaque burden compared to saline controls. Quantitative RT-PCR and western blot analysis revealed dKO mice had increased global and intraplaque polarization of T cells and macrophages to pro-inflammatory phenotypes, and also significantly increased TNFa expression in spleen and aortic arch compared to Ldlr-/- controls. Results from bone marrow transplantation experiments suggest immune cells participate in IL-19 mediated atheroprotection. Bone marrow derived macrophages (BMDMs) and vascular smooth muscle cells (VSMCs) isolated from dKO mice had significantly greater expression of TNFa mRNA and protein compared to controls. Importantly from a mechanistic standpoint, spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stability protein Human antigen R (HuR). BMDMs and VSMCs isolated from dKO mice also had greater HuR abundance. HuR stabilizes pro-inflammatory transcripts by binding AU-rich elements (AREs) in the 3’ untranslated region (UTR). Cytokine and HuR mRNA stability were increased in dKO BMDMs and VSMCs compared to controls, which was rescued by addition of IL-19 to these cells. IL-19 induces expression of miR133a, which targets and reduces HuR abundance; miR133a levels were lower in dKO mice compared to controls. Conclusions: These data indicate that IL-19 is an atheroprotective cytokine that decreases abundance of HuR, leading to reduced inflammatory mRNA stability. / Biomedical Sciences

Pontifícia Universidade Católica do Rio de Janeiro / Diabetes mellitus e doenças periodontais são altamente prevalentes na população mundial. Doenças periodontais (DPs) compreendem um grupo de condições crônicas inflamatórias induzidas por microorganismos que levam à inflamação gengival, à destruição tecidual periodontal e à perda óssea alveolar. Diabetes mellitus (DM) é o termo utilizado para descrever um grupo de desordens metabólicas associadas à intolerância à glicose e ao metabolismo inadequado de carboidratos. Uma vez que DPs poderiam agir de forma similar a outros estados infecciosos sistêmicos, aumentando a severidade do diabetes, uma possível relação entre ambas tem sido considerada em todo o mundo. Polimorfismos genéticos de um único nucleotídeo (SNPs) têm sido estudados em diversas doenças. Nas periodontites, acredita-se que possam estar envolvidos na exacerbação da resposta inflamatória frente ao desafio bacteriano, modificando a susceptibilidade do hospedeiro. Neste estudo, a prevalência de periodontite foi avaliada em portadores de diabetes mellitus tipo I. Posteriormente, o SNP localizado na região promotora do gene TNFA (-1031T>C) foi analisado e sua importância para a doença periodontal destrutiva foi avaliada. O grupo teste foi constituído por diabéticos tipo I (DGT, n=113) enquanto o grupo controle por indivíduos não diabéticos (ND, n=73). Para as análises dos polimorfismos genéticos, um subgrupo foi retirado do grupo teste (DG, n=58) e comparado ao grupo ND. Os seguintes parâmetros clínicos e demográficos foram avaliados: percentual de sítios com profundidade de bolsa &#61619; 6,0 mm (%PBS&#61619;6,0 mm); índice gengival (IG); perda óssea radiográfica (POR); fumo; duração do diabetes ; idade; índice de massa corpórea (IMC), n&#61616; de internações e n&#61616; de dentes presentes. Amostras de sangue e/ou esfregaço bucal foram colhidas de 58 pacientes do grupo teste e de 73 controles. Após a extração do DNA genômico e amplificação da região genômica de interesse por PCR (Polymerase Chain Reaction), o polimorfismo TNFA 1031T>C foi analisado por BbsI RFLP (Restriction Fragment Length Polymorphism). A análise dos produtos de digestão foi feita por eletroforese em gel de poliacrilamida 8%. A análise estatística das freqüências alélica e genotípica juntamente com os dados clínicos e epidemiológicos entre os 2 grupos foi feita através do teste do Mann-Whitney e do Qui-quadrado. Os grupos de estudo obedecem ao princípio de Hardy-Weinberg. No grupo ND, as seguintes freqüências genotípicas foram encontradas: 78,1% (T/T); 20,5% (T/C) e 1,4% (C/C) enquanto no grupo D foram: 42,4%(T/T); 37,3% (T/C) e 20,3% (C/C). A frequência do alelo T no grupo diabético (D) foi de 0,610 ao passo que no grupo ND foi de 0,883. Não foi possível encontrar uma relação entre o polimorfismo -1031 T>C do gene TNFA e a presença de periodontite em diabéticos tipo I. Entretanto, o polimorfismo estudado se mostrou significativamente relacionado (p<0,0001 e OR= 4.85 95%IC 2,271-10,338) à presença do diabetes tipo I. / Diabetes mellitus and periodontal diseases are highly prevalent worldwide. Periodontal disease (PD) combines a group of chronic inflammatory conditions induced by microorganisms that leads to gingival inflammation, periodontal tissue destruction and alveolar bone loss. Diabetes mellitus (DM) is a term used to describe a group of metabolic disorders associated to glucose intolerance and inappropriate carbohydrate metabolism. Once PDs could act in a similar way to other systemic infectious states increasing diabetes severity, a possible correlation between both has been considerate around the world. Single nucleotide polymorphisms (SNPs) have been studied in relation to several diseases. In periodontitis, it is believed to be involved in an exacerbated inflammatory response due to bacterial challenge, modifying the host susceptibility. In this study, the prevalence of periodontal disease was evaluated in type I diabetes patients (TIDM). Further on, the SNP in the promoter region of the TNFA gene (-1031T>C) was analyzed and its importance to destructive periodontal disease was considered. The test group was formed by type I diabetic patients (TDG, n=113) and the control group was composed by non-diabetic patients (ND, n=73). For the genetic polymorphism analysis a subgroup was taken from the test group (DG, n=58) and compared to the ND group. The following clinical and demographic parameters were assessed: percentage of sites with periodontal pocket depth (PPD) &#61619; 6.0mm; gingival index (GI); radiographic bone loss (RBL); smoking habits; diabetes duration; age; bone mass index (BMI); number of admissions and number of teeth. Blood samples and oral epithelial cells were collected from 58 patients of the test group and 73 controls. After genomic DNA extraction and amplification of the genomic region of interest by PCR (Polymerase Chain Reaction), the TNFA 1031T>C polymorphism was analyzed by BbsI RFLP (Restriction Fragment Length Polymorphism). Analysis of the digestion products were performed by 8% polyacrylamide gel electrophoresis. The statistical analysis of the genotypes and allelic frequencies along with the clinical and epidemiological data between the two groups were done through the Mann-Whitney and Chi-square tests. The study groups are in Hardy-Weinberg equilibrium. In the ND group, the following genotypic frequencies were found: 78.1% (T/T); 20.5% (T/C) e 1.4% (C/C), while for the D group, they were: 42.4% (T/T); 37.3% (T/C) e 20.3% (C/C). The frequencies of T allele in the D and ND groups were 0.610 and 0.883, respectively. It was not possible to find any correlation between the -1031 T>C promoter polymorphism of the TNFA gene and the presence of periodontitis in type I diabetic patients. However the studied polymorphism showed to be significantly related (p<0.0001 and OR=4.85 95%CI 2.271-10.338) to the presence of type I diabetes mellitus.

Pontifícia Universidade Católica do Rio de Janeiro / Diabetes mellitus e doenças periodontais são altamente prevalentes na população mundial. Doenças periodontais (DPs) compreendem um grupo de condições crônicas inflamatórias induzidas por microorganismos que levam à inflamação gengival, à destruição tecidual periodontal e à perda óssea alveolar. Diabetes mellitus (DM) é o termo utilizado para descrever um grupo de desordens metabólicas associadas à intolerância à glicose e ao metabolismo inadequado de carboidratos. Uma vez que DPs poderiam agir de forma similar a outros estados infecciosos sistêmicos, aumentando a severidade do diabetes, uma possível relação entre ambas tem sido considerada em todo o mundo. Polimorfismos genéticos de um único nucleotídeo (SNPs) têm sido estudados em diversas doenças. Nas periodontites, acredita-se que possam estar envolvidos na exacerbação da resposta inflamatória frente ao desafio bacteriano, modificando a susceptibilidade do hospedeiro. Neste estudo, a prevalência de periodontite foi avaliada em portadores de diabetes mellitus tipo I. Posteriormente, o SNP localizado na região promotora do gene TNFA (-1031T>C) foi analisado e sua importância para a doença periodontal destrutiva foi avaliada. O grupo teste foi constituído por diabéticos tipo I (DGT, n=113) enquanto o grupo controle por indivíduos não diabéticos (ND, n=73). Para as análises dos polimorfismos genéticos, um subgrupo foi retirado do grupo teste (DG, n=58) e comparado ao grupo ND. Os seguintes parâmetros clínicos e demográficos foram avaliados: percentual de sítios com profundidade de bolsa &#61619; 6,0 mm (%PBS&#61619;6,0 mm); índice gengival (IG); perda óssea radiográfica (POR); fumo; duração do diabetes ; idade; índice de massa corpórea (IMC), n&#61616; de internações e n&#61616; de dentes presentes. Amostras de sangue e/ou esfregaço bucal foram colhidas de 58 pacientes do grupo teste e de 73 controles. Após a extração do DNA genômico e amplificação da região genômica de interesse por PCR (Polymerase Chain Reaction), o polimorfismo TNFA 1031T>C foi analisado por BbsI RFLP (Restriction Fragment Length Polymorphism). A análise dos produtos de digestão foi feita por eletroforese em gel de poliacrilamida 8%. A análise estatística das freqüências alélica e genotípica juntamente com os dados clínicos e epidemiológicos entre os 2 grupos foi feita através do teste do Mann-Whitney e do Qui-quadrado. Os grupos de estudo obedecem ao princípio de Hardy-Weinberg. No grupo ND, as seguintes freqüências genotípicas foram encontradas: 78,1% (T/T); 20,5% (T/C) e 1,4% (C/C) enquanto no grupo D foram: 42,4%(T/T); 37,3% (T/C) e 20,3% (C/C). A frequência do alelo T no grupo diabético (D) foi de 0,610 ao passo que no grupo ND foi de 0,883. Não foi possível encontrar uma relação entre o polimorfismo -1031 T>C do gene TNFA e a presença de periodontite em diabéticos tipo I. Entretanto, o polimorfismo estudado se mostrou significativamente relacionado (p<0,0001 e OR= 4.85 95%IC 2,271-10,338) à presença do diabetes tipo I. / Diabetes mellitus and periodontal diseases are highly prevalent worldwide. Periodontal disease (PD) combines a group of chronic inflammatory conditions induced by microorganisms that leads to gingival inflammation, periodontal tissue destruction and alveolar bone loss. Diabetes mellitus (DM) is a term used to describe a group of metabolic disorders associated to glucose intolerance and inappropriate carbohydrate metabolism. Once PDs could act in a similar way to other systemic infectious states increasing diabetes severity, a possible correlation between both has been considerate around the world. Single nucleotide polymorphisms (SNPs) have been studied in relation to several diseases. In periodontitis, it is believed to be involved in an exacerbated inflammatory response due to bacterial challenge, modifying the host susceptibility. In this study, the prevalence of periodontal disease was evaluated in type I diabetes patients (TIDM). Further on, the SNP in the promoter region of the TNFA gene (-1031T>C) was analyzed and its importance to destructive periodontal disease was considered. The test group was formed by type I diabetic patients (TDG, n=113) and the control group was composed by non-diabetic patients (ND, n=73). For the genetic polymorphism analysis a subgroup was taken from the test group (DG, n=58) and compared to the ND group. The following clinical and demographic parameters were assessed: percentage of sites with periodontal pocket depth (PPD) &#61619; 6.0mm; gingival index (GI); radiographic bone loss (RBL); smoking habits; diabetes duration; age; bone mass index (BMI); number of admissions and number of teeth. Blood samples and oral epithelial cells were collected from 58 patients of the test group and 73 controls. After genomic DNA extraction and amplification of the genomic region of interest by PCR (Polymerase Chain Reaction), the TNFA 1031T>C polymorphism was analyzed by BbsI RFLP (Restriction Fragment Length Polymorphism). Analysis of the digestion products were performed by 8% polyacrylamide gel electrophoresis. The statistical analysis of the genotypes and allelic frequencies along with the clinical and epidemiological data between the two groups were done through the Mann-Whitney and Chi-square tests. The study groups are in Hardy-Weinberg equilibrium. In the ND group, the following genotypic frequencies were found: 78.1% (T/T); 20.5% (T/C) e 1.4% (C/C), while for the D group, they were: 42.4% (T/T); 37.3% (T/C) e 20.3% (C/C). The frequencies of T allele in the D and ND groups were 0.610 and 0.883, respectively. It was not possible to find any correlation between the -1031 T>C promoter polymorphism of the TNFA gene and the presence of periodontitis in type I diabetic patients. However the studied polymorphism showed to be significantly related (p<0.0001 and OR=4.85 95%CI 2.271-10.338) to the presence of type I diabetes mellitus.

Une des difficultés majeures dans le traitement des cancers est l’acquisition de résistance par les cellules tumorales vis-à-vis de la mort induite par les différentes chimiothérapies. Au sein du laboratoire, nous nous intéressons aux propriétés anti-tumorales d’un donneur de monoxyde d’azote (NO), le Glycéryl TriNitrate (GTN), fréquemment utilisé dans le traitement de l’angine de poitrine. Au cours de ce travail, nous avons étudié les mécanismes moléculaires par lesquels le GTN sensibilise les cellules tumorales de plusieurs types de cancer (colique, mammaire, prostatique) à la mort impliquant des voies de signalisation régulées par des cytokines telles que le TNFα, l’IL-6 ou encore le GDF-15.Une meilleure compréhension des mécanismes sous-jacents à l’action anti-tumorale du GTN permettrait de potentialiser son utilisation comme nouvelle thérapie anti-cancéreuse.Modèle colique : le GTN, en présence de la cytokine pro-inflammatoire TNFα, sensibilise les cellules cancéreuses coliques et mammaires à l’apoptose. Du point de vue mécanistique, le GTN induit la S-nitrosylation de cIAP1, inhibant ainsi son activité ubiquitine E3 ligase. Ce qui abroge la voie de signalisation classique NF-кB de survie cellulaire activée par la voie TNFα/TNFR1 en faveur d’une voie de signalisation pro-apoptotique.Modèle mammaire : le GTN intervient au niveau de la migration cellulaire en altérant la voie de signalisation Jak2/STAT3 activée par la cytokine pro-inflammatoire IL-6, dans un modèle de cancer du sein triple négatif. En présence de dérivés du platine (carboplatine) générant de l’IL-6, le GTN freine la migration des cellules en induisant la S-nitrosylation, et probablement l’inactivation, de la kinase Jak-2, indispensable pour l’activation de la voie.Modèle prostatique : le GTN sensibilise à la mort les cellules cancéreuses prostatiques résistantes au docétaxel en modulant le taux de deux marqueurs de résistance à cette chimiothérapie : la clusterine (CLU) et le growth differentiation factor 15 (GDF-15). Au niveau moléculaire, le GTN diminue le taux de l'isoforme cytoprotectrice soluble de la CLU (sCLU) et augmente le taux de l'isoforme cytotoxique nucléaire (nCLU) dans les cellules prostatiques résistantes au docétaxel. Plus particulièrement, en présence de GTN, nous avons établi un lien entre le GDF-15 et la modulation du taux des isoformes de la CLU. / One of the main difficulties in the treatment of cancers is the acquisition of resistance by the tumor cells vis-à-vis the death induced by the different chemotherapies. In the laboratory, we are interested in the anti-tumor properties of a nitric oxide (NO) donor, Glyceryl TriNitrate (GTN), frequently used in the treatment of angina pectoris. In this work, we investigated the molecular mechanisms by which GTN sensitizes tumor cells of several types of cancer (colonic, mammary, prostate) to death involving signaling pathways regulated by cytokines such as TNFα, IL-6 or GDF-15.A better understanding of the mechanisms underlying the GTN's anti-tumor action would make it possible to use it as a new anti-cancer therapy.Colon model: GTN, in the presence of the pro-inflammatory cytokine TNFα, sensitizes colon and mammary cancer cells to apoptosis. From a mechanistic point of view, GTN induces S-nitrosylation of cIAP1, thus inhibiting its ubiquitin E3 ligase activity. This abrogates the classical NF-κB signaling pathway of TNFα / TNFR1 activated cell survival in favor of a pro-apoptotic signaling pathway.Mammary model: GTN intervenes at the level of cell migration by altering the Jak2 / STAT3 signaling pathway activated by the pro-inflammatory cytokine IL-6, in a model of triple negative breast cancer. In the presence of platinum (carboplatin) derivatives generating IL-6, GTN inhibits cell migration by inducing S-nitrosylation, and probably inactivation, of Jak-2 kinase, essential for the activation of the way.Prostate model: GTN sensitizes prostatic prostate cancer cells to death by modulating the level of two markers of resistance to this chemotherapy: clusterin (CLU) and growth differentiation factor 15 (GDF-15). At the molecular level, GTN decreases the level of the soluble cytoprotective isoform of CLU (sCLU) and increases the level of nuclear cytotoxic isoform (nCLU) in prostatic cells resistant to docetaxel. More particularly, in the presence of GTN, we have established a link between GDF-15 and the modulation of the isoform rate of the CLU.

Dendritic cells (DCs) are a migratory group of bone-marrow-derived leukocytes specialized for uptake, transport, processing and presentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce DC maturation characterized by cytokine production, up-regulation of co-stimulatory molecules and an increased ability to activate T cells. DCs have the ability to restrict growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires' disease, in murine ER-derived organelles (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in the transition from innate to adaptive immunity (145). The primary polyphenol in green tea extract is the catechin (-)-epigallocatechin-3-gallate (EGCG) which accounts for most of the numerous reported biological effects of green tea catechins, including anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an important marker of mouse DCs, and surface expression of co-stimulatory molecules CD40, CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Treatment of the cells with EGCG inhibited the microbial antigen induced up-regulation of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dose dependent manner, induced production of the Th1 helper cell activating cytokine, IL-12, and the chemokines RANTES, MIP1a, and MCP-1. However, EGCG upregulated TNFa production. In addition, EGCG inhibited both Lp and LPS induced expression of both TLR2 and TLR4 as well as LPS-induced NF-kB activation; all of which are important mediators of DC maturation. The modulation of phenotype and function of DCs by EGCG has implications for host interaction with microbial pathogens like Lp, which involve TLR interaction.

Introduction Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder that encompasses two major subtypes; Crohn’s Disease (CD) and Ulcerative Colitis (UC). Our knowledge regarding disease pathogesis is rapidly increasing. However, these disease entities provide challenges in diagnosis, monitoring of disease activity and assessing individual response to treatment, because there is a lack of validated clinical biomarkers. Metabolomics involves the study of numerous analytes that have very diverse physical and chemical properties and occur in a wide concentration range. Early evidence suggests there is potential for metabolomic profiling to be used in the differentiation of CD and UC. However, knowledge is limited regarding the metabolic changes seen in relation to disease activity or to medical or surgical treatments. Aims A metabolomics approach was taken to determine whether metabolomic profiles could distinguish between patients with CD or UC and healthy controls. We also aimed to define the relationship between metabolomic profile and disease activity, and to determine the effect of medical (anti-TNFa agents) and surgical treatment on the metabolome. Methods A metabolomics approach was undertaken. Serum and urine sample sets were collected from a total of 41 patients with ulcerative colitis, 43 patients with Crohn’s disease, and 62 healthy controls (HC). In order to allow a comparison of metablomic profile and disease activity, 4 sample sets were taken from the same patient at 3 monthly intervals over the period of one year. Those patients undergoing either surgical or biological treatment had sample sets taken pre and post intervention. Metabolomic analysis using gas chromatography time of flight mass spectrometry (GC-ToF-MS) and ultra-high performance liquid chromatography Fourier Transform mass spectrometry (UHPLC-FTMS) was carried out on both serum and urine. Results Serum and urine GC-ToF-MS and UHPLC-FTMS metabolomic analyses show differentiation between UC, CD and healthy controls, most significantly in urine analyses. No significant differentiation was seen in pre- and post-surgical patients, or pre- and post-biological therapy patients. It was possible to differentiate surgical patients from healthy controls, especially in the urine analyses. Metabolite identification revealed consistently more dietary variation in the healthy controls than in the IBD patients. Significant differences (p < 0.05) were seen between healthy controls and IBD patients in classes of metabolites relating to the citric acid cycle and the uronic acid pathway, as well as amino acids, fatty acids and cholesterols. The behaviour or location of disease, or the disease activity score did not appear to influence the metabolome in either serum or urine analyses using GC-ToF-MS and UHPLC-FTMS. Conclusion Metabolomic profiling of urine and serum in IBD may provide a novel methodology aiding both clinical diagnosis through biomarker development, and advancing knowledge of disease pathogenesis.

The interaction between epithelial cells and macrophages is integral to mucosal immune fate: determining the decision between tolerance and immune activation/inflammation. Endotoxin tolerisation (ET) is a circumstance where cells go through a hypo-responsive state, unable to respond to further endotoxin-LPS challenge. Mucosal macrophages (MΦs) have a dual functionality that determines tolerance to commensal organisms or immune response to entropathogens such as E. coli. In the case of mucosal inflammatory pathologies, such as Crohn’s disease, this state of tolerance is broken, resulting in destruction of gut mucosal tissue where the macrophage phenotype has been altered from a regulatory M2-like subset phenotype to an inflammatory M1-like subset phenotype, responding to both pathogenic and commensal bacteria. Chronic inflammation by bacterial pathogen related molecular patterns (PAMPs), such as LPS, is well established to induce tolerisation. The aims of this project were firstly, to characterise the control of macrophage differentiation in a mucosal setting by investigating the immunomodulatory effects of PAMPs, such as LPS in presence or absence of TNFα and to investigate ET mechanisms associated with MΦ subsets responding to the entropathogen E. coli K12-LPS. Secondly, to investigate the effect of epithelial cells on macrophage subsets behaviour upon inflammation and ET. M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively, whereas differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. A transwell co-culture system of Caco2 cells and MΦ subsets was developed to mimic the cell-to-cell cross-talk between epithelial cells and immune cells. Mono- and co-culture models were pre-treated with either LPS, TNFα or IL-1β prior to stimulation by PAMPs. TNFα, IL-1β, IL-18, IL-6 and IL-10 were qualified by ELISA. Cytokines, PRRs and endogenous negative regulatory molecules were detected by RT-PCR and WB and epithelial barrier function was measured by trans epithelial electrical resistance (TEER). ET induced by K12-LPS failed to demonstrate a differential subset-specific response in MΦ mono-culture system whereas, LPS differentially suppress LPS induced cytokine expression in MΦ co-culture system. Tolerised M1- and M2-like MΦs exhibited a significant reduction in expression and secretion of pro-inflammatory cytokines and comparable levels of anti-inflammatory cytokine, IL-10. The suppression of pro-inflammatory cytokine in these MΦs appeared to be linked to the differential TLR4 expression and up-regulation of negative regulators, such as IRAK-M and Tollip. In addition, MΦ subsets differentially responded to inflammation induced by pro-inflammatory cytokines, TNFα and IL-1β in mono- and co-culture models. In conclusion, tolerisation induced in MΦs is presented by the suppression of pro-inflammatory cytokine, which is associated with corresponding up-regulation of IL-10, TLR4 receptor and the negative regulators, in a subset-independent manner. In the case of cross-talk between epithelial cells and macrophages however, a differential sensitivities to ET was displayed. These findings allow more understanding of MΦ subsets functions and ET mechanisms, which may be beneficial for the development of in-vitro models of MΦ subsets and therapeutics targeting Crohn’s diseases.