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Interactions between the gut microbiota, epithelial cells and dendritic cells

Louise Hjerrild Zeuthen  


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Accepted by: BioCentrum Technical University of Denmark
Defended on: December 19, 2007
Official opponents: Mogens Claesson , Agnes Wold , Alexandr Parlesak
Tutors: Hanne Frøkiær

Published in the PhD Database: January 16, 2008


English abstract
The mammalian intestinal tract is colonised by 3-500 different species of commensal bacteria existing in an overall symbiotic relationship with the host. The gut microbiota is pivotal for the development and maintenance of intestinal immunological homeostasis. It protects the host from colonisation by pathogens, enhances the development of the intestinal epithelium and contributes to host metabolisms. Clinical trials and studies form animal models have proven that the microbiota (mainly strains of bifidobacteria, lactobacilli and commensal Escherichia coli) is an important player in the pathogenesis of inflammatory bowel diseases and allergic diseases. This has brought focus on a new therapeutic avenue: modification of the intestinal flora by dietary supplementation with ¿probiotics¿. However, general claims regarding probiotic modulation of host immunity overstate our current understanding of their specific effects on molecular and cellular components of the immune system.
This thesis focuses on the role played by dendritic cells (DC) in recognition of the microbiota, and on the crosstalk between commensals, DC and intestinal epithelial cells (IEC). It contains a thorough introduction to the intestinal immune system, the composition and development of the microbiota, the molecular and cellular interactions between them, and three in vitro studies. The first study demonstrates that while strains of bifidobacteria all induce a tolerogenic maturation pattern in human DC, strains of lactobacilli induce divergent maturation patterns with some strains inducing high levels of Th1 skewing cytokines. Weak Th1 skewing lactic acid bacteria (LAB) abrogate the Th1-skewed maturation induced by potent Th1 skewing lactobacilli. Additionally, commensal strains of E. coli and Klebsiella pneumoniae act in synergy with LAB in inducing a Th1 skewed maturation pattern in DC.
The second study shows, that only gram-negative commensals can mature basolaterally located DC through an intact epithelial layer dependently on IEC-expressed Toll-like receptor (TLR)4. However, LAB abrogate this maturation dependently on IEC-expressed TLR2. The mere presence of IEC spent media changes the commensal-induced maturation pattern in DC to a less Th1 skewed phenotype. Thymic stromal lymphopoietin and transforming growth factor-beta are partly responsible for these effects but other IEC-secreted soluble mediators are likely to be involved. These findings suggest that IEC play an active role in maintaining intestinal homeostasis.
The third in vitro study assesses how DC utilise TLR2 and NOD2 in the recognition of lipoprotein and peptidoglycan derived from LAB and show that triggering of the two receptors mediates divergent responses. In DC derived from TLR2 deficient mice, the anti-inflammatory response towards bifidobacteria is abrogated and is instead Th1 skewed, and the inhibitory effect is lacking. In DC derived from NOD2 deficient mice the inhibitory effects of bifidobacteria persist and the overall cytokine response is less Th1 skewed.
Overall, these findings contribute to our understanding of how microbiota-derived components influence the most important player in maintenance of immunological tolerance towards the flora and hence maintenance of gut homeostasis ¿ namely the DC.



Danish abstract
Tarmen er koloniseret med 3-500 forskellige specier af commensale bakterier, som eksisterer i et symbiotisk forhold med værten. Tarmfloraen er vigtig for udvikling og vedligeholdelse af immunologisk homeostase i tarmen. Den beskytter værten mod kolonisering af patogene bakterier, fremmer udviklingen af epitelcellelaget og bidrager til værtens metabolisme. Kliniske studier og studier i dyremodeller har vist, at tarmfloraen spiller en vigtig rolle i patogenesen af inflammatoriske tarmsygdomme og allergiske sygdomme. Det har ført til øget fokus på en ny terapeutisk mulighed: modifikation af tarmfloraen ved indtagelse af ¿probiotika¿. Der er adskillige generelle påstande omkring gavnlige immunologiske effekter af probiotika, hovedsageligt af bifidobakterier, lactobacilli, og commensale Escherichia coli stammer, men deres specifikke effekter på molekylære og cellulære komponenter af immunsystemet er ringe belyst.
Denne afhandling fokuserer på dendritiske cellers (DC) rolle i genkendelsen af tarmfloraen og på kommunikationen mellem commensale bakterier, DC og tarmepitelceller. Først introduceres tarmens immunsystem, sammensætningen og udviklingen af tarmfloraen og molekylære og cellulære interaktioner mellem dem. Dernæst præsenteres tre in vitro studier. Det første studie viser, at mens bifidobakterier inducerer et tolerogent modningsmønster i humane DC, inducerer lactobacilli stammer forskellige modningsmønstre, hvor nogle stammer inducerer stor produktion af Th1-drejende cytokiner. Svage Th1-drejende mælkesyrebakterier hæmmer det Th1-drejede respons induceret af visse lactobacilli stammer. Commensale gram-negative bakterier, E. coli and Klebsiella pneumoniae stammer, inducerer et Th1-drejet modningsmønster i synergi med mælkesyrebakterier.
I det andet studie undersøges, hvorvidt commensale bakterier kan modne basolaterale DC gennem et epitelcellelag og viser, at kun gram-negative commensale bakterier kan modne underliggende DC afhængig af Toll-like receptor (TLR)4 udtrykt på epitelcellerne. Mælkesyrebakterier kan hæmme denne modning vha. TLR2 udtrykt på epitelcellerne. Vækstmedie fra epitelceller ændrer DC fænotypen til et mindre Th1-drejet modningsmønster i respons til commensale bakterier. Thymic stromal lymphopoietin og transforming growth factor-beta spiller en rolle, men det udelukker ikke at andre epitelcelle-afledte faktorer kan være involveret. Disse resultater tyder på, at epitelceller aktivt bidrager til opretholdelsen af homeostase i tarmen.
I det tredje in vitro studie undersøges, om TLR2 and NOD2 er involveret i DC genkendelse af lipoprotein og peptidoglycan fra mælkesyrebakterier, og det viser, at aktiveringen af de to receptorer medierer modsatrettede effekter. I DC afledt fra TLR2 knockout mus er immunresponset mod mælkesyrebakterier Th1-drejet og bifidobakterier kan ikke inhibere den Th1-drejende effekt af lactobacilli. I DC afledt fra NOD2 knockout mus er den inhibitoriske effekt af bifidobakterier upåvirket, men cytokinproduktionen er mindre Th1-drejet. Alt i alt bidrager disse resultater til forståelsen af, hvordan tarmflora-afledte komponenter påvirker DC, som er den vigtigste celletype i vedligeholdelse af immunologisk tolerance mod floraen og dermed vedligeholdelse af homeostasen.