Research into the gut microbiome has revealed the widespread influence that microbial species have on their host. Host genetics and environmental factors influence the abundance and diversity of the bacterial species living within the gastrointestinal tract. When the normal composition of the gut microbiota is altered, a dysbiotic state incurs. Inflammatory bowel disease (IBD) is a chronic/relapsing inflammatory disorder of the intestinal mucosa, which is characterized by a state of dysbiosis. Despite the large amount of information studying the role dysbiosis has in the pathogenesis of IBD, it is not clear how the altered microbial composition of the gut in IBD patients leads to susceptibility to enteric pathogens such as Clostridium difficile. This study aims to highlight the features of the gastrointestinal tract that are modified as a result of dysbiosis in the IBD population, and how these features facilitate colonization by C. difficile and symptom development. Review of the available literature demonstrated that the depletion of Clostridial cluster XIVa in IBD-associated dysbiosis alters bile acid metabolism and butyrate fermentation in the colon, ultimately promoting germination of C. difficile spores and weakening the gut's immune response against toxin-mediated inflammation. From continued research into the gut microbiota, more will be understood of how these microbial organisms influence human health and disease.

BACKGROUND: Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory conditions of the bowel that display a rising prevalence in childhood and adolescence. The diagnosis of a chronic condition, such as IBD, in childhood can be overwhelming and stressful for both the patient and caregiver. Parents and family members can play a critical role in providing emotional support for children with newly diagnosed IBD. We hypothesized that dysfunctional patient and parental coping strategies would correlate with increased anxiety and depression in children, worsening clinical disease activity, and increased healthcare utilization. OBJECTIVE: The primary objective of the IBD Coping Study is to assess the stability of coping strategies and psychological stress over the first year following a new diagnosis of IBD. Secondarily, we aim to assess the impact of child and parental coping strategies on disease activity and emotional well-being over the year. METHODS: This is a prospective, longitudinal cohort study of children with newly diagnosed IBD and their parents at Boston Children's Hospital (BCH). Patients between the ages of 9 and 17 years old that have been diagnosed with CD, UC or Indeterminate colitis (IC) within the last 6 months, are English-speaking, and receive routine care at BCH are approached for participation in our study. Participation includes the completion of previously validated psychological metrics for both child and parent at baseline and then again 12 months later. Our instruments include the Children's Depression Inventory (CDI), the Screen for Child Anxiety Related Disorders (SCARED), the IMPACT-III Questionnaire, the Patient Health Questionnaire (PHQ-9), Healthcare Utilization Survey, Pediatric Inventory for Parents (PIP), and the Hospital Anxiety and Depression Scale (HADS). RESULTS: We screened 187 patients with IBD for participation in our study, and roughly 30% of them were eligible for recruitment. To date, we have enrolled a total of 30 patients. Of these patients, there was an equal distribution of male and female participants. The majority of patients were around 14 years of age at the time of IBD diagnosis with a greater number of patients with CD (17) currently represented. We are approaching patients on average about 1.5 months after their initial diagnosis. The baseline average Pediatric Ulcerative Colitis Activity Index (PUCAI) score was 21.15 ± 20.53, whereas the average Pediatric Crohn's Disease Activity Index (PCDAI) score was 3.75 ± 2.50. On CDI items, teenage girls and boys reported increased raw and standardized scores (Raw: 5.83 and 4.83, respectively; Standardized: 43.67 and 44.67, respectively) than their younger counterparts for depressive behaviors, including negative mood and interpersonal problems. Pediatric patients encountered as inpatients reported an overall lower quality of life on IMPACT-III items (103.29 ± 15.11) than those approached in the ambulatory setting (140.36 ± 7.50). On SCARED items, patients met criteria for the potential presence of one or more anxiety disorders. Inpatients also reported being bothered more frequently with respect to hindrances in their sleep, appetite, and daily routines on the PHQ-9 metric. Parents of children with newly diagnosed IBD rely on increased communication with their child's primary GI provider, and their scores reflected lower emotional functioning during an admission period when compared to scores reported during regular scheduled ambulatory visits. Scores collected from the HADS screen demonstrate that 6% and 33% of parents reported a score great enough to be considered a "borderline case" for depression and anxiety measures, respectively. Primary comparisons between child health assessments and parent healthcare utilization depicted concurrent elevations in the same child-parent pair at baseline. CONCLUSION: Our initial findings suggest a clear disparity between emotional stability in children and their parents in outpatient and inpatient settings following a new IBD diagnosis. Healthcare utilization by parents may be linked to adaptive or maladaptive coping, and continuation of our study will substantiate this prediction. In looking ahead, potential interventions may require approaches stratified by age, gender, and hospital setting. Our study supports the need for further investigations into the impact of targeted interventions that promote an improvement in overall quality of life in children with IBD and their family during the first year of post-diagnosis.

Defensinas são peptídeos antimicrobianos produzidos na mucosa intestinal e fazem parte da imunidade inata, agindo sobre vários microrganismos luminais. Deficiência na expressão de defensinas tem sido relatada em doenças inflamatórias intestinais (DII), no entanto a contribuição de cada tipo de defensina, num cenário de polimorfismo genético, mantém alguma controversa. Βeta-defensinas humanas (HBDs) têm atividade antimicrobiana contra uma ampla variedade de fungos, bactérias e vírus e têm também, um papel na ligação entre a imunidade inata e adaptativa atuando como quimiotáticos. O gene DEFB1 (8p23), codificando a beta-defensina humana 1 (HBD-1), é expresso normalmente por células epiteliais de uma série de tecidos, mas sua expressão pode variar entre indivíduos e pode ser modificada durante processo inflamatório. Produção deficiente de defensinas parece contribuir para a patogênese de DII, e uma diminuição na expressão de HBD-1 tem sido relatada na mucosa de pacientes com doença de Crohn (DC) e retocolite ulcerativa (RCU). Nós avaliamos a possível associação de três polimorfismos do gene DEFB1 com a suscetibilidade a DII, RCU e DC, em 149 pacientes, 79 com DC e 70 com RCU; e 200 controles saudáveis do sul do Brasil. No nosso estudo não se observou diferença estatisticamente significativa entre a distribuição das frequências alélicas para DEFB1 SNPs -52G>A. -44C>G e -20G>A entre o total de pacientes com DII e controles. Porém, quando pacientes com DC foram estratificados de acordo com a localização anatômica, o alelo -20G>A foi mais frequente em pacientes com DC colônica do que em controles (65 % VS 44 %, p=0,048). De forma similar, o genótipo A/A foi mais frequente em pacientes com DC colônica do que em controles (36 % VS 16 %), mas neste caso, a diferença não foi estatisticamente significativa (p=0,07). Embora não se achou uma clara e forte associação entre os SNPs 5’-UTR DEFB1 e suscetibilidade/proteção à doença inflamatória intestinal, nossos resultados sugerem possível envolvimento do gene DEFB1 nestas enfermidades, especialmente com a localização colônica da doença de Crohn. Estudos com amostras maiores e populações diversas serão úteis para avaliar a tendência observada no nosso grupo. / Defensins are antimicrobial peptides produced by the intestinal mucosa and are part of the innate immune system, playing a protective role against various intestinal microorganisms. Deficiency in the expression of defensins has been reported in inflammatory bowel diseases (IBD), however there is some controversy over the contribution of each type of defensine, in a setting of great genetic polymorphism. Beta-defensins (HBDs) have an antimicrobial activity against a great variety of fungi, bacteria and viruses, and also have a role in connecting the innate and the adaptive immunity, acting as a chemostatic agent. Deficient production of defensins appears to contribute to the pathogenesis of IBD, and the lower expression of HBD-1 has been reported on the mucosa of Ulcerative colitis (UC) and Crohn’s disease (CD) patients. We evaluated a possible association of three polymorphisms of gene DEFB1 with susceptibility to develop IBD, UC and CD in 149 patients, 79 with CD and 70 with UC; and 200 healthy controls from the south of Brazil. The gene DEFB1 (8p23), which codifies human beta-defensin 1 (HBD-1), is constitutivelly expressed by epithelial cells of several tissues, but its expression may vary among different individuals and may be modified by inflammation. In our study we did not find a statistically significant difference between the distribution of the allelic frequencies for DEFB1 SNPs -52G>A, -44C.G and -20G>A between the total number of patients and controls. However, when patients were stratified according to the anatomic location, the allele -20G>A was more frequent in patients with colonic CD than in contros (65% VS 44%, p=0,048). Similarly, the genotype A/A was more frequent in patients with colonic CD than in controls (36% vs 16%), however, in this case, the difference wasn’t statistically significant (p=0,07). Although we did not find a clear and strong association between the 5’-UTR DEFB1 SNP and susceptibility to IBD, our results suggest a possible involvement of the DEFB1 gene and these diseases, particularlly colonic CD. Further studies with larger samples and diverse populations will be usefull to evaluate the trend observed by our group.

Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T19:23:38Z No. of bitstreams: 1 Micheli Stéfani Zarzecki.pdf: 2027524 bytes, checksum: 535aae8d63b5c3ca4ac4debb8ecb1f6f (MD5) / Made available in DSpace on 2016-04-18T19:23:38Z (GMT). No. of bitstreams: 1 Micheli Stéfani Zarzecki.pdf: 2027524 bytes, checksum: 535aae8d63b5c3ca4ac4debb8ecb1f6f (MD5) Previous issue date: 2016-02-19 / A Doença Inflamatória Intestinal (DII) é uma doença crônica, recidiva e de etiologia desconhecida. Fatores ambientais, estresse oxidativo e fatores imunológicos podem estar relacionados as causas dessa doença, e a colite ulcerativa e a doença de Crohn são exemplos dessa patologia. O mecanismo patogênico da DII é assumido como sendo um desequilíbrio da resposta imunológica a antígenos no ambiente intestinal. Os principais tratamentos das DII não são geralmente bem tolerados já que demonstram causar efeitos colaterais e, além disso, apresentam alta taxa de reincidências. A DII induzida por ácido 2,4,6-trinitrobenzeno sulfônico (TNBS) em modelos de roedores é caracterizada por aumento dos níveis de marcadores inflamatórios como TNF-α e interleucinas (IL-1β, IL-12, IL-17, IL-18 e IL-6). Tendo em vista a busca por alternativas eficazes nos tratamentos destas DII, os compostos orgânicos de selênio vêm se destacando como substâncias com potencial terapêutico, devido às suas atividades farmacológicas, como anti-inflamatória e antinociceptiva. Aliado a isso, o estudo de compostos de selênio combinados à oxiesteróis tem evidenciado resultados promissores. Demonstrou-se recentemente que o composto p-cloro-fenil-selenoesterol possui efeito antioxidante e anti-inflamatório em um modelo de dor e inflamação em camundongos. O objetivo desse trabalho foi investigar o efeito anti-inflamatório do composto p-cloro-fenil-viii selenoesterol (PCS), no modelo de DII induzida pelo TNBS em camundongos Swiss fêmeas. Os camundongos receberam o composto PCS) (10mg/kg; p.o.) durante todo o experimento (9 dias) por via oral. No quinto dia induziu-se a colite utilizando 2 mg de TNBS dissolvido em 0,1mL de uma solução de etanol a 50%, o qual foi administrado pela via intrarretal. No décimo dia, os camundongos foram eutanasiados e as amostras de sangue e cólon foram coletadas para as respectivas dosagens dos marcadores inflamatórios, de estresse oxidativo, analise histopatológica e sinais clínicos da doença. Os resultados obtidos demonstram que os animais tratados com TNBS apresentaram redução do comprimento colônico e aumento nos níveis da citocina pró inflamatória interleucina 6 (IL-6) e fator de necrose tumoral (TNF-α), e aumento dos níveis da enzima mieloperoxidase (MPO), tratamento com PCS foi capaz de reduzir os níveis das citocinas pró inflamatórias IL-6 e do TNF-α aos níveis do controle, além de evitar a redução do comprimento colônico que é um dos sinais da colite experimental induzida por TNBS. Nesse estudo também se observou uma melhora nos danos histológicos nos animais tratados com PCS em comparação com o grupo induzido por TNBS. Além disso, o tratamento com o PCS foi capaz de diminuir o estresse oxidativo e prevenir a diminuição das defesas antioxidantes no cólon de animais com DII induzida por TNBS. Portanto, nossos resultados sugerem que o tratamento com PCS apresentou uma melhora no quadro clínico da DII experimental em camundongos e que pode, futuramente e após mais estudos sobre este composto, tornar-se um potencial agente terapêutico para o tratamento de doenças inflamatórias, bem como as intestinais, colite ulcerativa e doença de Crohn. / The Inflammatory Bowel Disease (IBD) is a chronic, recurrent disease and of unknown etiology. Factors that may relate the causes of this disease are environmental factors, oxidative stress and immune factors. Example of this disease is the Ulcerative Colitis and Crohn's Disease. The main treatments of IBD are not generally well tolerated, they have side effects and have a high rate of relapse. The pathogenesis of IBD is assumed to be an imbalance of the immune response to antigens in the intestinal environment. IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rodent models is characterized by increased levels of inflammatory markers such as TNF-α and interleukins (IL-1β, IL-12, IL-17, IL- 18 and IL-6). The organic selenium compounds have been highlighted as substances with therapeutic potential because of their pharmacological activities such as anti-inflammatory and antinociceptive. Allied to this, the study combined with the selenium compounds oxiesteróis has shown promising results, it was demonstrated recently that PCS compound has antioxidant and anti-inflammatory effect in a model of pain and inflammation in mice. The aim of this study was to investigate the anti-inflammatory effect of p-chloro-phenyl-selenoesterol compound in IBD model induced by TNBS in Swiss female mice. The mice received the p-chlorophenyl-selenoesterol compound (PCS) (10mg / kg; po) throughout the experiment (9 days) orally. On the fifth day colitis was induced using 2 mg of TNBS dissolved in 0.1 mL of a 50% ethanol x solution, which was administered via intrarectal. On the tenth day, mice were euthanized and samples of blood and colon were collected for the respective levels of inflammatory markers of oxidative stress, histopathological and clinical signs of disease. The results show that animals treated with TNBS showed a reduction in colonic length, and increased levels of interleukin pro-inflammatory cytokine-6 (IL-6) and tumor necrosis factor (TNF-α) and increased levels of Myeloperoxidase enzyme (MPO), treatment with PCS was able to reduce the levels of pro inflammatory cytokines IL-6 and TNF-α levels to control and avoids the reduction in colonic length which is one of signs of experimental colitis induced by TNBS. In this study there was also an improvement in the histological analysis the animals treated with PCS compared with the group induced by TNBS. Furthermore, combined with improvements in inflammatory and histological parameters, treatment with PCS was able to decrease oxidative stress and prevent the decrease of antioxidant defenses in the colon of animals with TNBS-induced IBD. This finding suggests that treatment with PCS showed an improvement of the clinical picture in IBD in experimental mice and which could be a potential therapeutic agent for the treatment of inflammatory diseases, and intestinal, Ulcerative Colitis and Crohn's Disease.

Background The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) are chronic relapsing inflammatory disorders which have a rising incidence and cause significant morbidity. There are currently several treatment options with many more in the drug pipeline, but there are a lack of accurate biomarkers for decisions on treatment choice, assessment of disease activity and prognostication. There is a growing interest and desire for personalised or 'precision' medicine in IBD where novel biomarkers may help individualise IBD care in terms of diagnosis, choice of therapy, monitoring of response and detection of relapse. One class of functionally active biomarkers which have yet to be thoroughly investigated in IBD is damage-associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA). It has been recently shown that gut mitochondrial dysfunction can result in loss of epithelial barrier function and the development of colitis. Mitochondrial DAMPs have recently been described as elevated in several inflammatory diseases. Hypothesis The primary hypothesis of this thesis is that circulating levels of mtDNA is elevated in IBD. Secondary hypotheses are: (a) levels of other mitochondrial DAMPs are elevated in IBD, (b) circulating mtDNA can be used as a novel biomarker in IBD and (c) mtDNA is released locally at sites of inflammation in IBD. Methods Plasma and serum were collected prospectively from recruited IBD patients and non-IBD controls. Faeces and colonic tissue were collected from a subset of these patients. mtDNA in serum, plasma and faeces was measured using qPCR (amplifying COXIII/ND2 genes). Mass spectrometry was used to detect mitochondrial formylated peptides in the plasma of a subset of patients. IBD tissue was assessed for (a) mitochondrial damage using transmission electron microscopy (TEM) and (b) TLR9 expression, the target for mtDNA. Results 97 patients with IBD (67 UC and 30 CD), and 40 non-IBD controls were recruited. Plasma mtDNA levels were increased in UC and CD (both p < 0.0001) compared to non-IBD controls; with significant correlations with blood (CRP, albumin, white cell count), clinical and endoscopic markers of severity; and disease activity. In active UC, we detected significantly higher circulating mitochondrial formylated peptides and faecal mtDNA levels (vs. non-IBD controls [p < 0.01 and < 0.0001 respectively]) with demonstrable TEM evidence of intestinal mucosal mitochondrial damage. In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC/CD compared to controls (both p < 0.05). Conclusions Taken together, the findings suggest mtDNA is released during active inflammation in inflammatory bowel disease and is a potential novel mechanistic biomarker.

Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.

As Doenças inflamatórias intestinais (DII) são multifatoriais e sua etiologia envolve susceptibilidade genética, fatores ambientais, disbiose e ativação exacerbada do sistema imunológico no intestino. Essas doenças também tem sido relacionadas a baixos níveis de dehidroepiandrosterona (DHEA), um hormônio precursor de diversos esteroides e relacionado à modulação das respostas imunes. Porém, os mecanismos precisos que relacionam as ações deste hormônio com a proteção ou susceptibilidade à doença de Crohn ou colite ulcerativa ainda não são totalmente conhecidos. Sendo assim, este projeto buscou entender o papel imunomodulador do DHEA exógeno in vitro e in vivo durante a inflamação intestinal experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Inicialmente, in vitro, DHEA inibiu a proliferação de células do baço de forma dose dependente nas concentrações de 5?M, 50?M ou 100?M, com diminuição da produção de IFN-?. Este hormônio não foi tóxico para células de linhagem mieloide, embora tenha causado necrose em leucócitos nas doses mais elevada (50 ?M e 100?M), o que pode ter influenciado a diminuição das citocinas in vitro. Nos ensaios in vivo, os camundongos tratados com DHEA (40 mg/Kg) foram avaliados na fase de indução da doença (dia 6) e durante o reparo tecidual, quando os animais expostos ao DSS e ao DHEA por 9 dias foram mantidos na ausência destas drogas até o dia 15. Houve diminuição do escore pós-morte, melhora no peso e nos sinais clínicos da inflamação intestinal, com redução de monócitos no sangue periférico com 6 dias e aumento de neutrófilos circulantes na fase de reparo tecidual (15 dias). Ainda, a suplementação com DHEA levou à redução da celularidade da lâmina própria (LP) e ao restabelecimento do comprimento normal do intestino. O uso deste hormônio também diminuiu a expressão do RNAm de IL-6 e TGF-?, enquanto aumentou a expressão de IL-13 no colón dos animais durante a fase de indução da doença, o que provavelmente ajudou na atenuação da inflamação intestinal. Além disso, houve acúmulo de linfócitos CD4+ e CD8+ no baço e diminuição apenas de linfócitos CD4+ nos linfonodos mesentéricos (LNM), indicando retenção das células CD4+ no baço após uso do DHEA. O tratamento foi também capaz de aumentar a frequência de células CD4 produtoras de IL-4 e diminuir CD4+IFN-?+ no baço, além de reduzir a frequência de CD4+IL-17+ nos LNM, sugerindo efeito do DHEA no balanço das respostas Th1/Th2/Th17 relacionadas à colite. Em adição, as células de baço dos animais tratados com DHEA e expostos ao DSS se tornaram hiporresponsivas, como visto pela diminuição da proliferação após re-estímulos in vitro. Finalmente, DHEA foi capaz de atuar no metabolismo dos camundongos tratados, levando à diminuição de colesterol total e da fração LDL no soro durante a fase de indução da doença, sem gerar quaisquer disfunções hepáticas. Com isso, podemos concluir que o DHEA atua por meio do balanço das respostas imunes exacerbadas, minimizando os danos locais e sistêmicos causados pela inflamação intestinal induzida por DSS. / Inflammatory bowel diseases (IBD) are multifactorial diseases whose etiology involves genetic susceptibility, environmental factors, dysbiosis and exacerbated activation of the immune system in the gut. These diseases have also been associated to lower levels of dehydroepiandrosterone (DHEA), a precursor of various steroid hormones, related to modulation of immune responses. However, the precise mechanisms that link the actions of this hormone with protection or susceptibility to Crohn\'s disease or ulcerative colitis are still not fully understood. Thus, this project aimed to understand the immunomodulatory role of exogenous DHEA in vitro and in vivo in experimental intestinal inflammation induced by dextran sodium sulfate (DSS) in C57BL/6 mice. Initially, in vitro, DHEA inhibited the proliferation of spleen cells in a dose dependent way on the concentrations of 5?M, 50?M and 100?M, with decreased production of IFN-?. This hormone was not toxic to myeloid lineage cells, although it caused necrosis of leukocytes at the highest doses (50?M and 100?M), which may have influenced the decrease of the cytokines in vitro. Mice treated with DHEA (40 mg / kg) were evaluated at the induction phase of the disease (day 6) and during tissue repair, when animals exposed to DSS and DHEA for 9 days were maintained in the absence these drugs until the day 15. There was decrease of postmortem score, improved weight and clinical signs of intestinal inflammation, besides reduced peripheral blood monocytes on day 6, together with an increase in circulating neutrophils in tissue repair phase (15 days). Supplementation with DHEA also led to a reduction in cellularity of the lamina propria (LP) and to the restoration of normal length of the gut. The use of this hormone also decreased the expression of of IL-6 and TGF-? mRNA, while IL-13 was augmented in the colon of mice during the induction phase of the disease, a fact probably related to attenuation of intestinal inflammation. Furthermore, there was accumulation of CD4+ and CD8+ cells in the spleen along with decreased CD4+ leukocytes in mesenteric lymph nodes (MLN), indicating retention of CD4+ cells in the spleen after use of DHEA. The treatment was also able to increase the frequency of CD4+ cells producing IL-4 and decrease CD4+IFN-?+ in spleen, with reduced frequency of CD4+IL-17+ in the MLN, suggesting a role for DHEA on the balance of Th1/Th2/Th17 responses related colitis. In addition, splenocytes of mice treated with DHEA and exposed to DSS became hiporresponsives as seen by decreased proliferation after re-stimulation in vitro. Finally, DHEA was able to act on the metabolism of treated mice, leading to decreased total cholesterol and LDL cholesterol in serum during the induction phase of the disease, without generating any liver dysfunction. Thus, we concluded that DHEA acts by balancing the exacerbated immune responses, minimizing local and systemic damages caused by intestinal inflammation induced by DSS.