Pro-inflammatory cytokines released from immune cells infiltrating the endocrine pancreas in Type 1 Diabetes (T1D) induce the generation of reactive oxygen and nitrogen species (ROS/RNS). Cytokines are in part cytotoxic to ƓÒ-cells via the production of peroxynitrite (ONOO-). ƓÒ-cell are weakly protected against the toxicity of ROS/RNS because of limited expression of antioxidant enzymes. The purpose of this study was to evaluate the expression and regulation of Peroxiredoxins (Prdxs/PRDXs), a new family of antioxidant enzymes in islet ƓÒ-cell. Peroxiredoxin 5 (Prdx5) is ubiquitously expressed in mammals and it exhibits a range of cellular roles including cytoprotective antioxidant defence. Human PRDX5 possesses a peroxynitrite reductase activity but its role in ƓÒ-cell defence was not investigated yet. In a first set of experiments, the localization of the Prdx family was analyzed in rodent pancreas. Prdx1 was preferentially found in the non-b-cells of the islet and in exocrine tissue. Prdx2, Prdx3 and Prdx5 were present in b and non-b-cells, while Prdx4 and Prdx6 were poorly expressed. Then, we investigated the modulation of Prdx mRNA and protein expression levels by cytokines in adult rat isolated islets. Prdx1, Prdx2 and Prdx3 expression was not modified while Prdx5 mRNA was upregulated. However, Prdx5 protein was downregulated, which could involve ubiquitination and proteasomal degradation. Little is known about the PRDX antioxidant enzyme expression in human islets. In a second set of experiments, we investigated the expression and regulation of the 6 PRDXs in human islet preparations facing the context of T1D pathogenesis. PRDX 2, 3, 5, 6 were observed in the exocrine part of the pancreas. PRDX2 and PRDX6 were preferentially expressed in islet ƓÑ cells rather than in ƓÒ cells. PRDX3 and PRDX5 were localized in ƓÑ cells as well as in ƓÒ cells. PRDX4 was detected neither in exocrine nor in endocrine tissue. Islets exposed to a mixture of cytokines showed a downregulation of PRDX2, 3, 5, 6 mRNA expression, as was also the case for PRDX5 protein. This study demonstrated that a clear difference between human and rodent species does exist in terms of tissue localization, expression and regulation of Prdxs by cytokines. Finally, we performed Prdx5 overexpression or silencing in insulin secreting cell line INS-1E. Overexpression of Prdx5 was effective against a stress induced by SIN-1 but not against the cytokines mixture. On the opposite, silencing Prdx5 expression decreased the cell viability. Then, the hypothesis that the vulnerability of islets to cytokines mixture was due to the Prdx5 downregulation was not demonstrated. However, the modification of Prdx5 expression would in part be responsible for the high sensitivity of ƓÒ-cell to peroxynitrite. In conclusion, this study featured the presence of some Prdxs/PRDXs in islet cells, and the regulation of their expression by cytokines. They intervene in protection against ONOO- toxicity but their implication against cytokine agression remain to be more precisely evaluated.

Probiotics and prebiotics are emerging household terms, whose claimed health benefits share commonality. Their attributed health benefits include the production or induction of short chain fatty acids, maintaining bowel function, building colonization resistance (against pathogens) and treating antibiotic-associated diarrhea as well as colitis. Although both probiotic and prebiotic effects on immune system have been studied, the mechanisms of their activity are still not clearly defined and the conclusions drawn are elusive. While probiotics can act to influence the host at the cellular level, prebiotics, by definition, exert their effects indirectly through their impact on gut microbes. One purpose of this study was to investigate effects of Lactobacillus rhamnosus R0011 on innate immune parameters at the intestinal epithelial cell level, examining effects on both human and rat IEC. A second purpose was to define the effects of a range of prebiotic dietary fibres on the immune system at the mucosal and systemic level, using Biobreeding rats. L. rhamnosus demonstrated the ability to decrease proinflammatory cytokine and Toll-like receptor agonist-induced IL-8 and CINC-1 production from human and rat IEC, respectively. The timing of L. rhamnosus R0011 addition to HT-29 IEC, relative to proinflammatory challenge, influenced its ability to decrease IL-8 production. L. rhamnosus was more effective at decreasing production of IL-8 from human IEC when they were pre-incubated with this bacterium and subsequently challenged with proinflammatory stimuli. Certain effects of L. rhamnosus R011 were also observed in the absence of proinflammatory stimuli. Viable L. rhamnosus induced TNF-α production from rat IEC and heat-killed L. rhamnosus decreased constitutive TGF-β production from rat IEC and induced IL-8 or CINC-1 production from human and rat IEC, respectively. In Biobreeding rats, we demonstrated that oat dietary fibre significantly alters active TGF-β, CINC-1 and IL-6 levels in the colon in comparison to AIN-93G-fed rats. Wheat dietary fibre induced changes in active TGF-β, CINC-1 and IL-4 levels in the ileum in comparison to resistant starch-fed rats. Lastly, resistant starch exerted effects in the mesenteric lymph node, where changes in active TGF-β were observed in rats in comparison to AIN-93G-fed rats. Oat bran, wheat bran and resistant starch had no effects on cytokine levels in the serum or spleen of rats. Fructooligosaccharide-fed rats had a significant increase in active TGF-β levels in the colon and a significant decrease in active TGF-β levels in the spleen. Overall this suggests a FOS supplemented diet has both mucosal and systemic effects in rats, while wheat, oat and resistant starch supplemented diets had effects focused at the different locations at the mucosal level. These results illustrate differences in the ability of different dietary fibres to target immune parameters in specific mucosal tissues along the gastrointestinal tract and differential ability to exert systemic effects. Understanding the mechanism of action of probiotics provides insight into the downstream effects of prebiotics, while investigating effects of prebiotics on the immune system provides a broader view of the outcome of changes in gut microbiota composition and activity at the host organism level. / UOIT

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thèse doctorat : Médecine. Bioinformatique : Université de Nantes : 2005. / Bibliogr. 219-233 f. [308 réf.].

Thèse de doctorat : Pharmacologie : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 61-67).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 168-194). Also available in print.

During spermatogenesis, developing germ cells must migrate across the blood-testis barrier (BTB) and enter the adluminal compartment for further development. Throughout this process, extensive junction restructuring occurs at Sertoli-Sertoli and Sertoli-germ cell interfaces. Cytokines are known to play crucial roles in regulating testicular cell junction dynamics at different regulatory levels. However, the mechanism of cytokine-mediated regulation on newly identified junction molecules remains unclear. In this dissertation, the molecular mechanisms on how cytokines regulate the junction proteins of immunoglobulin superfamily (IgSF) including coxsackievirus and adenovirus receptor (CAR), nectin-like molecule-2 (Necl-2) and Necl-4 in testicular cells were studied. CAR is expressed on Sertoli and germ cells. It mediates both homophilic and heterophilic interaction for Sertoli-germ cell adhesion. It was found that combined treatment of interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα) reduced CAR mRNA and protein levels, and caused the disappearance of CAR from germ cell interface. IFN-γ+TNFα promoted CAR protein degradation via ubiquitin-proteasome pathway. In addition, IFN-γ+TNFα reduced CAR mRNA through regulating the binding of NF-κB subunits and SP/KLF proteins to CAR promoter. Collectively, these results demonstrated for the first time the potential mechanism utilized by IFN-γ+TNFα to exert their effects during testicular inflammation. Necl-2 is exclusively expressed by spermatogenic cells in the testis. In this study, it was demonstrated that transforming growth factor-β1 (TGF-β1) down-regulated Necl-2 mRNA and protein levels, and caused the disappearance of Necl-2 from cell surface. Using inhibitors and shRNAs, it was found that TGF-β1 induced Necl-2 protein degradation through clathrin-dependent endocytosis. Endocytosis assay further confirmed that TGF-β1 accelerated the internalization of Necl-2 to cytosol. Moreover, TGF-β1 repressed Necl-2 gene transcription in Smad-dependent manner. Taken together, these results unraveled the mechanism of how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring during spermatogenesis. Necl-4 has been detected in Sertoli cells, but little is known about its regulation in the testis. It was found that TNFα down-regulated Necl-4 mRNA and protein levels. Inhibitor studies suggested that both caveolin-dependent endocytosis and ubiquitin-proteasome pathway were involved in TNFα-induced Necl-4 protein degradation. Co-immunoprecipitation indicated that Necl-4 was physically associated with Par3, Par6, aPKC and Cdc42 which are the major components of polarity complex in mouse testis. Further study was shown that TNFα reduced the expression of Par3, and altered the binding between Necl-4 and Par complex in Sertoli cells. These results suggested that Necl-4-mediated cell adhesion could be disrupted by TNFα via reducing its expression and altering its interaction with Par complex. Studies reported herein suggest that junction proteins of the IgSF are precisely regulated by cytokines at transcriptional and post-translational levels. These results further enrich current understanding on how junction dynamics are regulated during spermatogenesis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Το ασβέστιο αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση του ενδοκυττάριου ασβεστίου αυξάνει την παραγωγή της IL-10 στα Τ-λεμφοκύτταρα, αλλά δεν είναι σαφές ποια ενδοκυττάρια μόρια εμπλέκονται σε αυτή τη ρύθμιση. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής του ασβεστίου στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα και η εντόπιση των πιθανών ενδοκυττάριων στόχων. Ο ρόλος των ενζύμων κλειδιών στην οδό ενεργοποίησης μέσω ασβεστίου καλσινευρίνης και CaM Kinase II μελετήθηκε με χρήση είτε ειδικών αναστολέων, κυκλοσπορίνης και ΚΝ-62 αντίστοιχα, είτε με πλασμίδια που κωδικοποιούν τις καταλυτικές υπομονάδες των ενζύμων καλσινευρίνη και CaM Kinase II. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28 (διέγερση μέσω TCR), ή Ionomycin/PMA (ενδοκυττάρια στόχευση Ca++ και PKC) παρουσία ή απουσία των ειδικών αναστολέων. Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10, IL-2, IL-4 παρουσία ή απουσία των ενεργών ενζύμων ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp Η δράση των ίδιων ενζύμων στην ενεργότητα των μεταγραφικών παραγόντων MEF2, CREB και NF-κB ελέγχθηκε με ταυτόχρονη έκφρασή τους in vivo με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο τους και in vitro σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA. Η επίδραση του μεταγραφικού παράγοντα MEF2 στον υποκινητή της IL-10 έγινε μέσω υπερέκφρασης του in vivo σε πειράματα διαμόλυνσης. Η IL-10 ανιχνεύεται 24 ώρες μετά τη διέγερση των κυττάρων, φτάνει στο μέγιστο στις 48 ώρες και μειώνεται μετά τις 72 ώρες. Παρουσία κυκλοσπορίνης η παραγωγή της IL-10 μειώθηκε κατά 80%-100%, ενώ παρουσία ΚΝ-62 η παραγωγή της IL-10 μειώθηκε κατά 70%-90%, σε όλα τα χρονικά διαστήματα στα οποία μελετήθηκε. Τα ποσοστά αναστoλής ήταν ίδια και κατά τους δύο τρόπους διέγερσης. Οταν τα κύτταρα διεγέρθηκαν μέσω TCR και του συνδιεγερτικού μορίου CD28, η αναστολή των παραπάνω ενζύμων δεν επηρεάζει την συσσώρευση του mRNA της IL-10, υποδηλώνοντας ότι η ρύθμιση γίνεται σε μεταγενέστερα της μεταγραφής στάδια, επηρεάζοντας πιθανά τη σταθερότητα του mRNA ή το ρυθμό μετάφρασής του. Όταν ο τρόπος διέγερσης των κυττάρων παρέκαμπτε τον TCR τότε η ελάττωση της παραγωγής της IL-10 αντανακλούσε και σε επίπεδο mRNA. Η ενεργοποίηση του υποκινητή της IL-10 που παρατηρείται μετά από διέγερση με ΙON/PMA και CD3/CD28 αυξήθηκε κατά 5 και 3 φορές αντίστοιχα σε Τ λεμφοκύτταρα τα οποία είχαν διαμολυνθεί με τις ενεργές μορφές των ενζύμων καλσινευρίνη και CaM Kinase II σε σχέση με τα κύτταρα τα οποία είχαν διαμολυνθεί μόνο με το όχημα pSRa. Η δράση της CaM Kinase II είναι εκλεκτική για τον υποκινητή της ΙL-10 εφόσον το ίδιο ένζυμο μειώνει την δραστικότητα του υποκινητή τόσο μιας άλλης Th2 κυτταροκίνης όπως η IL-4 όπως επίσης και της IL-2, ενώ η καλσινευρίνη έχει θετική επίδραση στους υποκινητές και των τριων κυτταροκινών. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από τα προαναφερθέντα ένζυμα καλσινευρίνη και CaM Kinase II βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2, CREB και NFκΒ όπως ελέγχθηκε με το πρόγραμμα Consite. Η in vitro δραστικότητα του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώθηκε κατά 50% παρουσία των αναστολέων της καλσινευρίνης και της CaM Kinase II ενώ η ενεργότητα του αυξήθηκε μετά από υπερέκφραση της CaM Kinase II. Αντίθετα η ενεργότητα τόσο του μεταγραφικού παράγοντα CREB όσο και του NFκB μειώθηκε παρουσία της CaM Kinase II, γεγονός που υποδεικνύει τον MEF2 ως ενδοκυττάριο στόχο της ασβεστιοεξαρτώμενης οδού ενεργοποίησης στη ρύθμιση της παραγωγής της IL-10 από ανθρώπινα T λεμφοκύτταρα. Το γεγονός ότι η υπερέκφραση του MEF2D σε Τ-λεμφοκύτταρα αύξησε την ενεργότητα του υποκινητή της IL-10 αποδεικνύει ότι είναι ένας σημαντικός παράγοντας στη ρύθμιση της παραγωγής της. / Calcium is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of calcium has been shown to increase IL-10 production. The aim of this study was the elucidation of the role of calcium in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of the specific inhibitors of calcineurin and CaM Kinase II, CsA and KN-62 respectively while there were used plasmids coding the catalytic subunits of calcineurin and CaM Kinase II. The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR. The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1327bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp).Similarly, we studied the activity of IL-2 and IL-4 promoter The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE The activity of MEF2 NF-kB and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors.. Binding of the transcription factor MEF2 was investigated in nuclear extracts of stimulated human T cells with EMSA experiments, whilw his effect on IL-10 promoter was determined through transfecrtion experiments, using a MEF2D plasmid. IL-10 production reaches its peak at 48 hours and it is diminished after 72 hours. We observed a 80-100% reduction of IL-10 production in the presence of CsA and a 70-90% reduction in the presence of KN-62. The inhibition was the same regardless the way of stimulation. When the cells are stimulated through TCR and costimulatory molecule CD28 the inhibition of calcineurin and CaM Kinase II does not affect mRNA accumulation, suggesting that the mechanism of regulation is post-transcriptional, possibly through changes in mRNA stability or through different translation rate, while when cells are Ion/PMA stimulated the reduction of IL-10 protein is also observed at the mRNA level. The induction of IL-10 promoter observed after Ion/PMA stimulation and CD3/CD28 is 3 and 5-fold increased in T cells transfected with the active forms of the enzymes calcineurin and CaM Kinase II. The effect of CaM Kinase II is selective only for the Il-10 promoter, as the same enzyme decreases the activity of IL-2 and IL-4 promoter, while Calcineurin has the same effect on all three promoters. The part of the promoter of IL-10 that shows to be affected by the previously mentioned enzymes is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. The binding of MEF2 to nuclear extracts of stimulated T cells is reduced by 50% in the presence of CsA and KN-62, while his activity is induced in t cells transfected with CaM Kinase II. On the contrary, CREB and NF-kB activities are reduced in the presence of CaM Kinase II, a fact indicating MEF2 as an impotant transcription factor for the regulation of IL-10 production. This is also shovn by the fact that overexpression of MEF2D in T cells increases the activity of IL-10 promoter.

Objective: Study the effects of vitamin D on viral driven innate immune responses, by looking at differences in cytokine production, receptor expression, and endogenous vitamin D levels. Methods: Primary peripheral blood mononuclear cells (PBMC) and epithelial cells (EC) were cultured in the presence of viral ligands and vitamin D. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) were used to determine cytokine production and mRNA expression. Results: PBMC stimulated with toll-like receptor 4 ligand (TLR4L), but not viral TLR8L, led to decreased pro- and anti-inflammatory cytokine production in the presence of 1,25(OH)2D3. RIG-like receptor (RLR) activation, on the other hand, in primary EC exhibited decreased pro-inflammatory cytokine production in the presence of vitamin D. Conclusions: Our findings are among the first to show differences between bacterial and viral driven innate immune responses in the presence of vitamin D. As responsiveness in RLR activated primary EC was altered in the presence of vitamin D, our data reveal the importance of studying the immune system as a whole.