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The damaged mucosal barrier in intestinal inflammation: interaction with therapeutic bacteria and roles for metallothioneins as immunomodulators
Book - Dissertation
De ziekte van Crohn en colitis ulcerosa zijn de belangrijkste chronische ontstekingsziekten van de darm. Recente studies benadrukken het belang van schade aan de epitheelbarrière en defecten in het aangeboren immuunsysteem in het ontstaan van beide ziekten. Deze schade zorgt ervoor dat de interactie tussen de microbiële darmflora en de gastheer niet meer optimaal verloopt. Het darmepitheel dient een fysische barrière te vormen tussen het lumen en de onderliggende mucosa. Bij de ziekte van Crohn en colitis ulcerosa worden afwijkingen in de mucuslaag, de integriteit en de permeabiliteit van het epitheel gevonden, waarvan men denkt dat ze een directe invloed hebben op de initiatie en de progressie van de ziekte. Naast zijn rol als fysische barrière heeft het epitheel ook een belangrijke taak in de identificatie van bacteriën en de gepaste reactie hierop. Het aangeboren immuunsysteem kan op twee manieren een rol spelen in the pathogenese. Een eerste theorie stelt dat defecten in het uitschakelen van proinflammatoire signalen een overdreven aangeboren immuunantwoord veroorzaken en een verlies van tolerantie tegenover de commensale microbiota. Volgens de tweede theorie worden pathogenen niet goed herkend, waardoor er een inefficiënte opruiming volgt die ervoor zorgt dat de exogene stimulus blijft bestaan en er een overdreven secundair immuunantwoord op gang komt. Het eerste deel van deze thesis handelt over de interactie van therapeutische bacteriën met de beschadigde epitheelbarrière. Genetisch gemodificeerde Lactococcus lactis bacteriën werden ontwikkeld om te zorgen voor een lokale toelevering van het anti-inflammatoir cytokine interleukine (IL)-10 in de mucosa. Aangezien deze lokale toelevering uitermate belangrijk is voor de therapeutische werking van de bacteriën, hebben we de interactie onderzocht tussen deze bacteriën en de darmmucosa. We vonden dat de bacteriën op transcellulaire wijze kunnen opgenomen worden in gezond follikelgeassocieerd epitheel. Onze data suggereerden eveneens opname door dendritische cellen, maar niet door M cellen. In ontstoken mucosa bemerkten we een verhoogde opname door paracellulair transport. Dit was waarschijnlijk veroorzaakt door de beschadigingen aanwezig in de mucosale barrière. We konden aantonen dat er levende en IL-10-producerende lactococcen in de lamina propria aanwezig waren. Uiteindelijk kon de afwezigheid van lactococcen in de mesenterische lymfeknopen en de milt uitsluiten dat deze bacteriën in de systemische circulatie terechtkomen. In het tweede deel onderzochten we de rol van metallothioneïnes (MTs) in darmontsteking. We vonden een significante daling in de mRNA en eiwitexpressie van MTs in het epitheel van gezond darmweefsel van patiënten met de ziekte van Crohn. Wanneer we bacteriële stimulaties uitvoerden op een darmepitheel cellijn met deficiënte MT expressie zagen we dat deze deficiëntie zorgde voor een daling in de secretie van het chemokine IL-8. Een gedetailleerde studie van dextraan sodiumsulfaat-colitis gaf aan dat MTnull muizen een zwaardere histologische inflammatie vertoonden dan wild type muizen in de vroege fase van colitis, een verschil dat te wijten was aan epitheliaal MT. Beide studies wijzen in de richting van een immunomodulatorische rol voor epitheliaal MT in het acute immuunantwoord van de darm. Deze verminderde basale epitheelexpressie vonden we niet terug bij colitis ulcerosa. In deze en andere ontstekingsziekten van de darm vonden we dat actieve inflammatie zorgde voor een daling in de epitheelexpressie van MT tegenover een stijging in de expressie van MT in de lamina propria. De ontstekingsafhankelijke daling in het epitheel komt niet overeen met de gekende inductie van MTs door proinflammatoire cytokines en reactieve zuurstofradicalen die geproduceerd worden tijdens ontsteking. Wij vonden dat transforming growth factor (TGF)-β de expressie van MT in epitheelcellen kon doen afnemen. In tegenstelling tot deze afname in het epitheel werd tijdens actieve inflammatie een toename in de expressie waargenomen in cellen van de lamina propria, voornamelijk veroorzaakt door fibroblasten van het granulatieweefsel die sterk positief waren. We suggereren dat MTs een cytoprotectieve rol hebben in deze cellen, wat een voordeel betekent in een inflammatoir milieu. Uiteindelijk hebben we een overzicht gemaakt van de mechanismen die de expressie van MTs regelen in ziekte en gezondheid.
Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, immunologically-mediated disorders of the intestine, the main forms being Crohn’s disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in the pathogenesis of IBD. These two components cause defects in the interaction of the host with the bacterial flora. The epithelium must maintain a physical barrier between the lumen and the underlying mucosa. Defects in the mucus layer, integrity, and permeability are found in IBD and are thought to have direct implications on disease initiation and/or progress. Furthermore, it has become clear that the intestinal epithelial cell lining exerts an important role in the identification of bacteria and the appropriate counteraction. The involvement of the innate immune system can be explained by two theories. The first one states that an exaggerated innate response and a loss of tolerance for the commensal intestinal microflora are caused by defective down-regulation of pattern recognition receptor signalling. The second theory encompasses a defective innate immunity originating from failure of recognition of bacterial threats or an ineffective response, which results in persistence of the exogenous stimulus and an exaggerated secondary immune response. The first part of this thesis focuses on the interaction of therapeutic bacteria with the damaged epithelial barrier. Genetically modified Lactococcus lactis bacteria were developed in order to provide a local mucosal delivery of the anti-inflammatory cytokine interleukin (IL)-10. Since local delivery of the cytokine in the tissue seems primordial, we focused on the in vivo interaction of the bacteria with intestinal murine mucosa and observed differences between an intact and an inflamed (damaged) epithelial barrier. In healthy intestinal tissue, we found an occasional transcellular uptake in follicle-associated epithelium. Our data were suggestive of dendritic cell sampling, but not of sampling by M cells. In inflamed mucosal tissue, both confocal and electron microscopic analysis suggested an enhanced uptake of lactococci through the paracellular route, which is likely to be a result of the damaged mucosal barrier. We were able to show the presence of viable and IL-10-producing lactococci in ileal and colonic lamina propria. The absence of lactococci in mesenteric lymph nodes or in the spleen excluded a systemic circulation of the bacteria, being safe for administration in patients. In the second part, the role of metallothioneins (MTs) as immunomodulators in intestinal inflammation is investigated. We found a significantly lower mRNA and protein expression in the epithelium of unaffected tissue of Crohn’s disease patients. In vitro studies with an MT-knockdown intestinal epithelial cell line showed a defective secretion of the neutrophil chemokine IL-8 upon bacterial challenge. A time course study of dextran sodium sulphate-induced colitis revealed that MTnull mice exhibited a significantly more severe histological inflammation in the early phase of colitis compared with wild type mice, a difference which was due to epithelial MTs. Both these in vitro and in vivo studies point to an immunomodulatory role for epithelial MTs in the acute intestinal immune response. In ulcerative colitis, we did not find this impaired basal epithelial MT expression. Here, active inflammation caused a down-regulation of epithelial MT and an up-regulation of non-epithelial MT which was also observed in other forms of intestinal inflammation. The inflammation-dependent down-regulation in the epithelium is in contrast with the known induction of MTs by proinflammatory cytokines and reactive oxygen intermediates, which are produced during inflammation. We found that transforming growth factor (TGF)-β, one of the few inhibitors identified for MT mRNA production, could decrease epithelial MT expression in ex vivo experiments. Contrary to the decrease in the epithelium, an increase in MT-expressing cells occurs in the lamina propria during active inflammation, predominantly caused by strongly MT-expressing fibroblasts of the granulation tissue. Since we did not find a correlation between MT expression and proliferation (a correlation which has been reported in vitro in the literature), we suggest that the role of MTs in these cells is a cytoprotective one, which would be beneficial for host cells in the inflammatory environment. Finally, we reviewed the MT regulatory mechanisms in health and in disease.
Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, immunologically-mediated disorders of the intestine, the main forms being Crohn’s disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in the pathogenesis of IBD. These two components cause defects in the interaction of the host with the bacterial flora. The epithelium must maintain a physical barrier between the lumen and the underlying mucosa. Defects in the mucus layer, integrity, and permeability are found in IBD and are thought to have direct implications on disease initiation and/or progress. Furthermore, it has become clear that the intestinal epithelial cell lining exerts an important role in the identification of bacteria and the appropriate counteraction. The involvement of the innate immune system can be explained by two theories. The first one states that an exaggerated innate response and a loss of tolerance for the commensal intestinal microflora are caused by defective down-regulation of pattern recognition receptor signalling. The second theory encompasses a defective innate immunity originating from failure of recognition of bacterial threats or an ineffective response, which results in persistence of the exogenous stimulus and an exaggerated secondary immune response. The first part of this thesis focuses on the interaction of therapeutic bacteria with the damaged epithelial barrier. Genetically modified Lactococcus lactis bacteria were developed in order to provide a local mucosal delivery of the anti-inflammatory cytokine interleukin (IL)-10. Since local delivery of the cytokine in the tissue seems primordial, we focused on the in vivo interaction of the bacteria with intestinal murine mucosa and observed differences between an intact and an inflamed (damaged) epithelial barrier. In healthy intestinal tissue, we found an occasional transcellular uptake in follicle-associated epithelium. Our data were suggestive of dendritic cell sampling, but not of sampling by M cells. In inflamed mucosal tissue, both confocal and electron microscopic analysis suggested an enhanced uptake of lactococci through the paracellular route, which is likely to be a result of the damaged mucosal barrier. We were able to show the presence of viable and IL-10-producing lactococci in ileal and colonic lamina propria. The absence of lactococci in mesenteric lymph nodes or in the spleen excluded a systemic circulation of the bacteria, being safe for administration in patients. In the second part, the role of metallothioneins (MTs) as immunomodulators in intestinal inflammation is investigated. We found a significantly lower mRNA and protein expression in the epithelium of unaffected tissue of Crohn’s disease patients. In vitro studies with an MT-knockdown intestinal epithelial cell line showed a defective secretion of the neutrophil chemokine IL-8 upon bacterial challenge. A time course study of dextran sodium sulphate-induced colitis revealed that MTnull mice exhibited a significantly more severe histological inflammation in the early phase of colitis compared with wild type mice, a difference which was due to epithelial MTs. Both these in vitro and in vivo studies point to an immunomodulatory role for epithelial MTs in the acute intestinal immune response. In ulcerative colitis, we did not find this impaired basal epithelial MT expression. Here, active inflammation caused a down-regulation of epithelial MT and an up-regulation of non-epithelial MT which was also observed in other forms of intestinal inflammation. The inflammation-dependent down-regulation in the epithelium is in contrast with the known induction of MTs by proinflammatory cytokines and reactive oxygen intermediates, which are produced during inflammation. We found that transforming growth factor (TGF)-β, one of the few inhibitors identified for MT mRNA production, could decrease epithelial MT expression in ex vivo experiments. Contrary to the decrease in the epithelium, an increase in MT-expressing cells occurs in the lamina propria during active inflammation, predominantly caused by strongly MT-expressing fibroblasts of the granulation tissue. Since we did not find a correlation between MT expression and proliferation (a correlation which has been reported in vitro in the literature), we suggest that the role of MTs in these cells is a cytoprotective one, which would be beneficial for host cells in the inflammatory environment. Finally, we reviewed the MT regulatory mechanisms in health and in disease.
Publication year:2009
Accessibility:Open