Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 hours post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78±0.88 and 13.90±3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93±0.28, 1.86±0.27, and 6.34±0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment, and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. In conclusion, this study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Aktuelle Studien legen nahe, dass ein niedriger Vitamin D-Status assoziiert mit steigenden Breitengraden, mit dem Auftreten von Autoantikörpern und damit einhergehenden Autoimmunerkrankungen positiv korreliert. Trifft UV-B-Strahlung auf die Haut, entsteht aus 7-Dehydrocholesterol nach enzymatischen Prozessen in Leber und Niere, die bioaktive Form von Vitamin D, 1α,25-Dihydroxyvitamin D3 (Calcitriol). Den finalen Stoffwechselschritt katalysiert die 1α-Hydroxylase CYP27B1. Die biologische Wirkung von Calcitriol wird über dessen Bindung an den nukleären Vitamin D-Rezeptor vermittelt, was letztlich zur Transkription der Zielgene führt. Die T-Zell-abhängige Sensibilisierung von Vitamin D-defizienten Cyp27b1-KO- und Wildtyp-Tieren mit Ovalbumin zeigte verstärkte humorale Immunantworten mit erhöhten Konzentrationen von Gesamt- sowie spezifischem IgG1, IgE und IgA bei den Cyp27b1-KO-Mäusen. Die Untersuchung der Leukopoese der Cyp27b1-KO-Tiere zeigte, dass die untersuchten lymphatischen Organe verminderte Gesamtzellzahlen gegenüber Wildtyp-Tieren aufwiesen. Die Präsenz und Verteilung in den jeweiligen Zellkompartimenten offenbarte keine wesentlichen Abweichungen zwischen Cyp27b1-KO- und Wildtyp-Mäusen. In einem Krankheitsmodell mit dem Helminthen Heligmosomoides polygyrus bakeri fielen die Cyp27b1-KO-Tiere durch dem Wildtyp gegenüber erhöhten Gesamt- sowie spezifischen IgE-Werten auf. Zwischen beiden Genotypen zeigten sich keine Unterschiede bei parasitologischen Parametern wie der Wurmlast, der Eianzahl pro Gramm Faeces sowie der Fekundität. Die Ergebnisse dieser Arbeit bestätigen, dass endogen-produziertes Vitamin D einen Einfluss auf die Funktionsweise von Lymphozyten hat. Dies äußert sich in verstärkten IgE-abhängigen Immunantworten bei Cyp27b1-KO-Tieren. In einem Parasiteninfektionsmodell wurden erneut verstärkte IgE-Antworten beobachtet, jedoch waren keine pathophysiologischen Konsequenzen in Bezug auf die Wurmabwehr nachweisbar. / Current studies demonstrate that low vitamin D levels associated with higher latitudes correlate with the occurrence of autoantibodies and linked diseases. Following UV-B radiation of the skin, numerous enzymatic reactions in liver and kidneys causes 7-dehydrocholesterol to turn into the bioactive 1α,25-dihydroxyvitamin D3 (calcitriol). The final and crucial step is thereby performed by the enzyme CYP27B1, an 1α-hydroxylase. The effect of calcitriol is mediated through binding to the vitamin D receptor, resulting in the transcription of target genes. T cell-dependent sensitization of Cyp27b1-wildtype and Cyp27b1-KO mice with ovalbumin revealed an increased humoral immune response in Cyp27b1-KO mice reflected by elevated concentrations of total and specific IgG1, IgE and IgA. Analysis of the leukopoiesis showed a diminished total cell count in bone marrow, thymus, spleen and peritoneal cavity in Cyp27b1-KO compared to Cyp27b1-wildtype mice. However, appearance and distribution of the analyzed cell compartments were comparable. A disease model using the intestinal nematode Heligmosomoides polygyrus bakeri demonstrated enhanced secretion of total and specific IgE in Cyp27b1-KO mice, which confirmed our previous findings. However, this showed no effect on parasite rejection, as seen in comparable results for worm burden, eggs per gram faeces and fecundity of female worms in Cyp27b1-wildtype and Cyp27b1-KO mice. Our work verified the role of endogenous vitamin D for lymphocyte development revealed by increased IgE-dependent immune responses in Cyp27b1-KO mice. Infection with H.p. bakeri confirmed enhanced IgE-responses, however, these results revealed no benefit in parasite clearance.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

<p>In hyperparathyroidism (HPT) raised serum concentrations of ionized calcium is caused by increased secretion of parathyroid hormone (PTH) by parathyroid tumors. Active vitamin D, 1α,25-dihydroxyvitamin D₃, is known to suppress PTH secretion and to reduce proliferation of parathyroid tumor cells.</p><p>The aim of this thesis was to examine expression of vitamin D hydroxylating enzymes, regulating the activation and inactivation of vitamin D and to study effects of vitamin D analogues, in parathyroid tumors and breast cancer.</p><p>The vitamin D activating enzyme, CYP27B1/25-hydroxyvitamin D₃ 1α-hydroxylase (1α-hydroxylase) and the vitamin D inactivating enzyme CYP24A1/25-hydroxyvitamin D₃ 24-hydroxylase (24-hydroxylase) were expressed in parathyroid tumors and breast cancer. </p><p>The parathyroid tumors had raised expression levels of 1α-hydroxylase and reduced levels of 24-hydroxylase in comparison to normal parathyroid glands, indicating ability for endogenous activation of vitamin D. The expression of 1α-hydroxylase may be of therapeutic advantage for local activation of non-1α-hydroxylated vitamin D analogues in tumor cells, thereby reducing unwanted hypercalcemic effects. </p><p>Three of five selected low calcemic vitamin D analogues had as efficient PTH suppressing effect, in bovine parathyroid cells, as three vitamin D analogues used clinically for treatment of secondary HPT.</p><p>The non-1α-hydroxylated vitamin D analogue EB1285 showed antiproliferative and PTH suppressive effects as well as transcriptional activity in parathyroid and breast tumor cells, respectively.</p><p>Ketoconazole, an inhibitor of vitamin D hydroxylating enzymes, suppressed PTH secretion and potentiated the effect of vitamin D analogues. Combined treatment with vitamin D analogues and specific 24-hydroxylase inhibitors may be important for future therapy. </p>

In hyperparathyroidism (HPT) raised serum concentrations of ionized calcium is caused by increased secretion of parathyroid hormone (PTH) by parathyroid tumors. Active vitamin D, 1α,25-dihydroxyvitamin D3, is known to suppress PTH secretion and to reduce proliferation of parathyroid tumor cells. The aim of this thesis was to examine expression of vitamin D hydroxylating enzymes, regulating the activation and inactivation of vitamin D and to study effects of vitamin D analogues, in parathyroid tumors and breast cancer. The vitamin D activating enzyme, CYP27B1/25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) and the vitamin D inactivating enzyme CYP24A1/25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) were expressed in parathyroid tumors and breast cancer. The parathyroid tumors had raised expression levels of 1α-hydroxylase and reduced levels of 24-hydroxylase in comparison to normal parathyroid glands, indicating ability for endogenous activation of vitamin D. The expression of 1α-hydroxylase may be of therapeutic advantage for local activation of non-1α-hydroxylated vitamin D analogues in tumor cells, thereby reducing unwanted hypercalcemic effects. Three of five selected low calcemic vitamin D analogues had as efficient PTH suppressing effect, in bovine parathyroid cells, as three vitamin D analogues used clinically for treatment of secondary HPT. The non-1α-hydroxylated vitamin D analogue EB1285 showed antiproliferative and PTH suppressive effects as well as transcriptional activity in parathyroid and breast tumor cells, respectively. Ketoconazole, an inhibitor of vitamin D hydroxylating enzymes, suppressed PTH secretion and potentiated the effect of vitamin D analogues. Combined treatment with vitamin D analogues and specific 24-hydroxylase inhibitors may be important for future therapy.

Epilobium hirsutum L. (Onagraceae) is a flowering, tall and perennial plant and native to Eurasia. It shows analgesic, anti-microbial and anti-proliferative activity, and it is used in our country as an alternative medicine. The pharmacological effect of Epilobium hirsutum L. could be explained by the presence of polyphenolics including steroids, tannins and flavonoids in the aerial parts. Viscum album L. (Loranthaceae) is a shrub that grows as an epiphyte on the branches of deciduous trees. It involves in the enhancement of macrophage phagocytic and cytotoxic mediated abilities as well as the strengthening the immune system. CYP24A1 and CYP27B1 are members of cytochrome P450 superfamily and the most important enzymes involved in the metabolism of vitamin D3. CYP27B1 and CYP24A1 are mitochondrial enzymes and also known as 25-hydroxyvitamin D3 1alpha-hydroxylase and 24-hydroxylase, respectively. CYP24A1 involves in 24-hydroxylation of 25-OH-D3 and 1,25-(OH)2D3 which is required for the catabolism of vitamin D3 compounds while CYP27B1 involves in 1&alpha / -hydroxylation of 25-OH-D3 into 1,25-(OH)2D3. In this study, in vivo effects of Epilobium hirsutum and Viscum album (subspecies growing on pine-trees-subsp. austriacum (Wiesb.) Vollmann) on rat liver CYP24A1 and CYP27B1 mRNA and protein expressions were investigated. To achieve this goal, 37.5 mg water extract of Epilobium hirsutum L./kg body weight/day was intraperitoneally injected to male rats for 9 days. To study the effect of Viscum album L., 10 mg water extract of Viscum album L./kg body weight/day was injected with the same conditions. After decapitation, livers were removed and S1.5 fractions were prepared. Effects of Epilobium hirsutum L. and Viscum album L. on rat liver mRNA and protein expressions were analyzed by qRT-PCR and western blotting, respectively. Epilobium hirsutum L. extract caused 31% and 18% decrease in rat liver CYP24A1 (p&lt / 0.0001) and CYP27B1 (p&lt / 0.05) protein expressions, respectively. The effect of Epilobium hirsutum L. on mRNA expression of CYP24A1 could not be observed, because CYP24A1 mRNA was almost undetectable in liver. Injection of Epilobium hirsutum L. to rats caused 2.7 fold increase in mRNA expression of CYP27B1 with respect to controls and normalized with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression as an internal reference (p&lt / 0.005). Viscum album L. caused 17% decrease in CYP24A1 protein expression (p&lt / 0.05). When rats injected with plant extract of Viscum album L., 18% decrease in CYP27B1 protein expression was observed (p&lt / 0.05). The effect of Viscum album L. on mRNA expression of CYP24A1 could not be observed since CYP24A1 mRNA was almost undetectable in liver. Injection of Viscum album L. to rats caused 3.8 fold increase in mRNA expression of CYP27B1 with respect to controls and normalized with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression as an internal reference (p&lt / 0.005). In conclusion, vitamin D3 metabolism may be affected by medicinal plants Epilobium hirsutum L. and Viscum album L. due to the changes in mRNA and protein expressions of CYP24A1 and CYP27B1 enzymes.